Derivatives of neuraminic acid

ABSTRACT

Provided are new derivatives of neuraminic acid of formula (I), where Ac represents an acyl residue of an aliphatic, araliphatic, aromatic, alicyclic, or heterocyclic carboxylic acid, including carboxylic amides, their 2-hydrocarbyl-glycosides, and their peracylated derivatives at the hydroxy groups of both these series of amides. These compositions are therapeutically useful in providing a protective effect against the neurotoxicity induced by excitatory amino acids, and can therefore be used in therapies of the central nervous system. ##STR1##

This is the U.S. national stage entry of PCT/US93/07307, filed Aug. 3,1993.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to new derivatives of neuraminic acid,especially carboxylic amides of the following formula, ##STR2## where Acrepresents an acyl residue of a carboxylic acid of the aliphatic,araliphatic, aromatic, alicyclic or heterocyclic series, comprising thecarboxylic amides, their 2-hydrocarbyl-glycosides, and their peracylatedderivatives at the hydroxy groups of both these series of amides.

These compounds have interesting pharmacological properties, especiallya protective effect against the neurotoxicity induced by excitatoryamino acids of the of glutamic acid type, and can therefore be used intherapies of the central nervous system, such as those followingcerebral degenerations or lesions, e.g., ischemia, hypoxia, epilepsy,trauma or compressions, metabolic dysfunctions, aging, toxic-infectiveand chronic neurodegenerative diseases, like Alzheimer's, Parkinson's,and Huntington's diseases.

The carboxylic amides and their derivatives of formula I according thepresent invention are new.

2. Description of Related Art

In the literature, there is a description of the non-substituted amideof N-acetyl-neuraminic acid, prepared as an intermediate in thesynthesis of tetrazolyl-2-decarboxy-N-acetyl-neuraminic acid (see Ann.1986, 2104-11).

In an article published in Hoppe Seyler's Physiol. Chemie, 1983, 364(109) 1411-17, there is a description of the amides obtainable throughthe reaction of the benzylketoside of N-acetyl-neuraminic acid withL-glycine, L-glutamic acid, and L-phenylalanine, followed by theelimination of the benzyl group through catalytic hydrogenation; nopharmacological action is described for these derivatives.

SUMMARY OF THE INVENTION

In addition to providing new derivatives of neuraminic acid, the presentinvention also provides pharmacological preparations containing theaforesaid derivatives for therapeutic use.

A third object of the present invention concerns the therapeutic use ofthese preparations.

A final object of the present invention concerns procedures for theproduction of these new derivatives.

Further scope of the applicability of the present invention will becomeapparent from the detailed description provided below. However, itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Even so,the following detailed description should not be construed to undulylimit the present invention, as modifications and variations in theembodiments herein discussed may be made by those of ordinary skill inthe art without departing from the spirit or scope of the presentinventive discovery.

The contents of each of the references cited herein are incorporated byreference in their entirety.

The amides and their derivatives according to the present invention canderive from both possible anomeric forms in position 2 of neuraminicacid, and therefore all the new compounds can be of either type at thatposition. The steric configuration of the other carbon atoms of theneuraminic residue is the same as that of the natural acid.

The acyl group Ac on the nitrogen of the neuraminic acid residue in theaforesaid formula has at least 4 and not more than 24 carbon atoms, andderives from non-substituted or substituted acids, preferably from 1 to3 functions selected from the group consisting of halogen atoms; free,esterified, or etherified hydroxylic or mercapto groups; free oresterified carboxylic or sulfonic groups, or such groups transformedinto amides; and free hydrocarbylic groups or hydrocarbylic groupssubstituted aminic groups.

These acids can be interrupted by --SO--, --SO₂ --, or phenylene groupsin the carbon atom chain of the hydrocarbylic residue. The halogen atomsare preferentially fluorine, bromine, or chlorine. Esterified hydroxylicor mercapto groups can derive from one of the acids mentioned regardingthe Ac group, but they preferentially derive from aliphatic or aromaticacids with not more than 8 carbon atoms. Moreover, they can derive frominorganic acids such as, for example, sulfuric or phosphoric acid, orespecially from their partial esters with mono- or polyvalent aliphaticalcohols, eventually with hydroxylic groups or aminic functionssubstituting in the hyrdocarbylic residues. Finally, they can derivefrom hydrocarbylsulfonic acids.

Etherified hydroxy or mercapto groups, or esterified carboxylic orsulfonic groups, preferentially derive from alcohols of the aliphaticseries having no more than 8 carbon atoms, or from the araliphaticseries with only one benzene ring and an alkylene of 1 or 2 carbonatoms. The hydrocarbylic groups which can substitute the aminic groupspreferentially derive from these alcohols; the amino groups can also bein the form of quaternary ammonium salts, e.g., tetraalkyl groups, e.g.,tetrabutylammonium.

Ac groups containing functionally modified hydroxy, mercapto, or aminogroups can also be present in the form of hydrocarbylic residues of theAc acyl group, interrupted in the carbon atom chain by the heteroatoms--O--, --S--, or --NH--, and, in the particular case of esters ofhydroxy or mercapto groups with partially esterified sulfuric orphosphoric acid, by groups of the type ##STR3## and in those of esterswith hydrocarbylsulfonic acids (e.g., p-toluenesulfonic ormethanesulfonic acid), by --O--SO₂.

The hydrocarbylic residue Ac, as already stated, can be blocked bysulfoxide or sulfonyl residues. In amides, converted carboxylic orsulfonic groups preferentially derive from lower aliphatic amines withnot more than 4 carbon atoms, or from araliphatic amines with only onebenzene ring and one or two carbon atoms in alkenyl residue.

The acids from which the Ac groups of the aliphatic series derive can hesaturated or unsaturated, and in this case, they preferentially haveonly one double bond, and can have linear or branched chains. Ofparticular interest are the following acids: butyric, valeric,particularly normal valeric and isovaleric, trimethylacetic (pivalicacid), caproic, isocaproic, enantic, caprylic, pelargonic, capric,undecilic, di-tert-butyl-acetic, 2-propyl-valeric (valproic acid),lauric, tridecilic, myristic, pentadecilic, palmitic, margaric, stearic,arachic, behenic, and lignoceric.

Among substituted aliphatic acids, levulinic acid must be mentioned;among dicarboxylic acids, succinic acid; and among natural amino acids,e.g., valine, leucine, phenylalanine, tryptophan, aminobutyric acid,methionine, lysine, aspartic acid, glutamic acid, proline,hydroxyproline; among the acids substituted with halogens, mono- anddichloroacetic acid, trichlorobutyric acid, and dibromobutyric acid.

The Ac group of formula I can also derive from natural or syntheticpeptides preferentially having not more than 12 amino acids, selectedfrom naturally occurring amino acids, e.g., those aforesaid.

Of particular interest according to the present invention are those inwhich Ac is an acyl residue belonging to peptides of the thymus gland.Acids, from which derive an Ac group of araliphatic nature, are e.g.,phenylacetic, cinnamic, phenylpropionic or atropic acid. Among aromaticacids, benzoic acid and its methylated homologues, salicylic acid,anthranilic acid, trimethoxybenzoic acid, phthalic or terephthalic acid,O,O'-dicarbonic acid, chlorobenzoic acid, vanillic acid, and veriatricor piperonilic acid must be mentioned.

Among Ac acyl groups belonging to acids of the alicyclic series, theremust be mentioned cyclohexane- and cyclopentane-carbonic acids,hexahydrophthalic, hexahydroisophhtalic and hexahydroterephthalic acids,camphoric and apocamphoric acid, and, among acids with a higher carbonatom content, prostaglandins and steroidic acids such as, for example,cholanic or cholic acid.

If Ac represents an acyl group belonging to an acid of the heterocyclicseries, this can be one of the following acids: nicotinic orisonicotinic, cinconninic, lysergic, isolysergic, dihydrolysergic,2-bromo-lysergic, 2-bromo-dihydrolysergic, 1-methyl-lysergic,1-methyl-dihydro-lysergic, 1-methyl-2-bromo-lysergic ortheophyllinacetic.

The carboxylamido functions according to the present invention canderive from ammonia (and in this case it is the non-substituted amide,--CONH₂), or from primary or secondary aliphatic, aromatic, araliphatic,alicyclic or heterocyclic amines, which can also be substituted in thehydrocarbylic residue by one to three functions selected from the groupconsisting of free, esterified, or etherified hydroxylic or mercaptogroups, halogens, free, esterified, or amide-modified carboxylic orsulfonic groups, and free or hydrocarbyl-substituted amino groups,wherein the hydrocarbyl group is blocked with an --SO-- or --SO₂ --group. These amines have no more than 24 carbon atoms.

The functions which can eventually substitute the carbon atom chain ofthe amide or the amine are preferentially those mentioned for the Acgroup of formula I. Aliphatic amines can have an open, saturated,unsaturated, linear, branched or cyclic chain. Of particular interestare alkyl- and dialkylamines having from 1 to 12 carbon atoms, such as,for example, methylamine, ethylamine, propylamine, hexylamine,diethylamine, dimethylamine, diisopropylamine, dihexylamine andalkylenylamines having from 3 to 6 cyclic carbon atoms, wherein therings are substituted or non-substituted, preferentially between one andthree C1-14 alkyl groups, e.g., methyl groups, like pyrrolidine,piperidine, and azepine.

The hydrocarbylic chains can also be blocked with heteroatoms such as,for example, --O--, --S--, or --NH-- groups, or they can be substituted,as already mentioned, with different functions, particularly alcoholic,amino, mercapto, carboxylic, and sulfonic functions, or by theirfunctionally modified forms, such as esters, ethers, or alkylatedderivatives. Of particular interest are the following: aliphaticdiamines, like ethylenediamine, trimethylenediamine,tetramethylenediamine, penta- and hexamethylenediamine, piperazine andits N-alkyl or C-alkyl derivatives having a C1-4 alkyl; aminoalcoholslike aminoethanol or aminopropanol; aminomercaptanes likemercaptoethylamine; aliphatic aminoacids like all those mentioned forthe Ac group of formula I; and aminosulfonic acids like taurine.Moreover, morpholine and thiomorpholine and their alkylated derivativessuch as, for example, those which are N- or C-methylated, are alsouseful.

The amide groups eventually can derive from peptides, such as thosementioned for the Ac group.

Of particular interest also are some derivatives of amines having alarge number of carbon atoms of an aliphatic nature, and which arerelated to phospholipids or sphingolipids or similar derivatives.

According to the present invention, the carboxylic group of theN-acyl-neuraminic acids can contain saturated or unsaturated aminicgroups having between 14 and 24 carbon atoms, or bases present in thefollowing lipids: phosphatidylethanolamine, phosphatidylserine,sphingosine, dihydrosphingosine, psychosine, dihydropsychosine,phosphorylcholine-sphingosine, phosphorylcholine-dihydrosphingosine, andphytosphingosine.

The carboxylamides of the present invention can also derive fromaromatic or araliphatic amines, but preferentially from those havingonly one aromatic ring, which can be substituted with 1 to 3 functionalgroups selected from the group consisting of halogens, hydroxylic ormethoxylic groups, carboxylic or sulfonic groups, or C1-4 loweraliphatic hydrocarbylic groups such as, for example, aniline,anthranilic acid, 1-amino-4-sulfonic acid, and benzylamide. In theaforesaid aliphatic amines, in one or more positions of thehydrocarbylic chain, a phenyl group can be present to block the carbonatom chain.

Amines which can be used for the conversion into amides according to thepresent invention include, for example, amines of pyrimidines such ascyanmethine, i.e., 2,4-dimethyl-6-amino-pyrimidine, purine derivativessuch as adenine, 4-aminouracil, and guanine, and alkaloids such asephedrine, tyramine, and adrenalin.

The 2-hydrocarbyl-glycosides of the aforesaid amides of neuraminic acidsof formula I derive from alcohols of the aliphatic, cycloaliphatic,aromatic, araliphatic or heterocyclic series, particularly from alcoholsof the aliphatic series having not more than 12 carbon atoms, or fromthe araliphatic series having preferentially only one benzene ring,eventually substituted with 1-3 lower C1-4 alkyl groups, for examplemethyl groups, and not more than 4 carbon atoms in the aliphatic chain,or from alcohols of the alicyclic or aliphatic-alicyclic series havingonly one cycloaliphatic ring and not more than 14 carbon atoms, or fromthe heterocyclic series having not more than 12, and especially 6 carbonatoms, and only one heterocyclic ring containing 1 or 2 heteroatomsselected from the group consisting of --NH--, --O-- and --S--. Thesealcohols can also be substituted, particularly with functions selectedfrom the group consisting of hydroxy, amino, and alkoxy groups havingnot more than 4 carbon atoms, and carboxylic and carbalkoxy groupshaving not more than 4 carbon atoms in the alkyl residues.

The aforesaid alcohols can be mono- and polyvalent, particularlybivalent. Among alcohols of the aliphatic series, of particular interestare lower alcohols having not more than 6 carbon atoms such as, forexample, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, andtert-butyl alcohol, and ethyleneglycol and propyleneglycol, amongdivalent alcohols. Among alcohols of the araliphatic series, ofparticular interest are those having only one benzene residue, likebenzyl and phenetyl alcohol; among alcohols of the alicyclic series,preferred are those having only one cycloaliphatic ring, likecyclohexylic alcohol. Among alcohols of the alicyclic series having morerings, steroid alcohols such as, for example, those of the pregnanegroup, like corticosteroids, and among these methylprednisolone, must bementioned. Among alcohols of the heterocyclic series there must bementioned tetrahydrofuranol, tetrahydropyranol, furfuryl alcohol andpyridylcarbinol.

In peracylated derivatives of the amides and their2-hydrocarbyl-glycosides, the hydroxy groups in position 2,4,7,8 and 9are acylated with acids belonging to the aliphatic, aromatic,araliphatic, alicyclic and heterocyclic series. Peracylated derivativesderive preferentially from acids of the aliphatic series having not morethan 10 carbon atoms, like formic, acetic, and butyric acid and theirisomers; valeric acids, like normal valeric, or pivalic acid; andcapronic or capric acid. These acids can also be substituted, and theperacylated derivatives can therefore derive from hydroxyacids likelactic acid, from amino acids like glycine, or from dibasic acids likesuccinic, malonic or maleic acid. Among aromatic acids, there must bementioned those with only one benzene ring, particularly benzoic acidand its derivatives with methyl, hydroxy, amino or carboxylic groupssuch as, for example, p-aminobenzoic, salicylic and phthalic acid.

The new compounds according to present invention can eventually betransformed into their acidic addition salts or into metallic salts withorganic bases, if the corresponding basic or acidic functions arepresent. These salts can also be used for the therapeutic purposesdescribed infra. With resepect to this equivalence between salts andamides in free form, it is obvious that what will be described for thecompounds in free form, especially their pharmaceutical and medicalapplications, is also true for the corresponding salts, provided thatthese salts are therapeutically acceptable, and therefore they also forman object of the present invention. These salts can also be used for thepurification of the amides, and in this case also, therapeuticallynon-acceptable bases and acids can be used, such as salts of picric andpicrolonic acid.

Compounds of the Present Invention

Defined compounds according to the present invention include the amide,methylamide, ethylamide, dimethylamide, diethylamide, propylamide,glycine amide, L-serine amide, aminobutyric amide, L-cysteine amide,taurine amide, the amide of cysteic acid, homocysteic acid,N-palmitoyl-neuraminic acid, N-stearoyl-neuraminic acid,N-acetyl-neuraminic acid, N-propionyl-neuraminic acid,N-pivaloyl-neuraminic acid, N-valeroyl-neuraminic acid,N-caproyl-neuraminic acid, N-lauroyl-neuraminic acid,N-succinyl-neuraminic acid, phenylacetyl-neuraminic acid,benzoyl-neuraminic acid, trimethoxy-benzoyl-neuraminic acid,phthaloyl-neuraminic acid, chlorobenzoyl-neuraminic acid,vanilloyl-neuraminic acid, cyclopenthane- andcyclohexane-carbonyl-neuraminic acid, N-nicotinyl-neuraminic acid,N-isonicotinyl-neuraminic acid, lisergyl-neuraminic acid,2-bromo-lisergyl-neuraminic acid, 1-methyl-lisergyl-neuraminic acid,theophillineacetyl-neuraminic acid, and their 2-glycosides derived fromone of the following alcohols: methyl alcohol, ethyl alcohol, propylalcohol, isopropyl alcohol, butyl alcohol, isobutyl alcohol, tertbutylalcohol, ethyleneglycol, propyleneglycol, benzyl alcohol,methylprednisolone, tetrhydrofuranol, tetrahydropyranol, furfurylalcohol, and pyridylcarbinol.

Other compounds of the present invention include the amide, methylamide,ethylamide, dimethylamide, diethylamide, propylamide, glycine amide,L-serine amide, aminobutyric amide, L-cysteine amide, taurine amide ofN-acylneuraminic acids having an acyl group deriving from one of thefollowing acids: aminobutyric, methionine, lysine, aspartic acid,glutamic acid, proline, tryptophan, or from an acyl residue derivingfrom a peptide present in the thymus, and their 2-glycosides derivingfrom one of the following alcohols: methyl alcohol, ethyl alcohol,propyl alcohol, isopropyl alcohol, butyl alcohol, isobutyl alcohol,tertbutyl alcohol, ethyleneglycol, propyleneglycol, benzyl alcohol,methyl-prednisolone, tetrahydrofuranol, tetrahydropyranol, furfurylalcohol, and pyridylcarbinol.

Another group of interesting compounds according to the presentinvention is formed by the amides deriving from pyrrolidine, piperidine,azepine, ethylenediamine, trimethylenediamine, hexamethylenediamine,piperazine or N-methyl or N-ethyl-piperazine, aminoethanol,aminopropanol, mercaptoethylamine, morpholine, tiomorpholine, orpeptides like those present in the thymus, and fromphosphatidylethanolamine, phosphatidylserine, sphingosine, psychosine,dihydropsychosine, sphingosylphosphorylcholine,dihydrosphingosylphosphorylcholine, or from the phytosphingosine of oneof the following N-acyl-neuraminic acids: N-palmitoyl-neuraminic acid,N-stearoyl-neuraminic acid, N-acetyl-neuraminic acid,N-propionyl-neuraminic acid, N-pivaloyl-neuraminic acid,N-valeroyl-neuraminic acid, N-caproyl-neuraminic acid,N-lauroyl-neuraminic acid, N-succinyl-neuraminic acid,phenylacetyl-neuraminic acid, phthaloyl-neuraminic acid,chlorobenzoyl-neuraminic acid, N-nicotinyl-neuraminic acid,N-isonicotinyl-neuraminic acid, lisergyl-neuraminic acid,2-bromo-lisergyl-neuraminic acid, 1-methyl-lisergyl-neuraminic acid,theophillineacetyl-neuraminic acid, and their 2-glycosides derived fromone of the following alcohols: methyl alcohol, ethyl alcohol, propylalcohol, isopropyl alcohol, butyl alcohol, isobutyl alcohol, tertbutylalcohol, ethyleneglycol, propyleneglycol, benzyl alcohol,methylprednisolone, tetrhydrofuranol, tetrahydropyranol, furfurylalcohol, and pyridylcarbinol.

Other derivatives are those peracylated at the hydroxy groups ofN-acyl-neuraminic acid, and particularly peracetates, perpropionates,perbutyrates, pervalerianates, perpivalates, persuccinates andperbenzoates.

Synthesis of the Compounds of the Present Invention

The present invention also comprises processes for the preparation ofthe new amides of N-acyl-neuraminic acids, their2-hydrocarbylglycosides, and their salts.

These processes are already known, and consist of the stepwiseintroduction of the amine function and eventually of the 2-glycosidicgroup into an N-acyl-neuraminic acid, and eventually of acyl groups intothe hydroxy groups and the final formation of their salts.

In a preferred embodiment, the carboxylic group of an N-acyl-neuraminicacid, in which the acyl group is desired in the final compound, or its2-hydrocarbyl-glycosidic derivatives, is transformed to an amide groupand, if desired, the 2-hydrocarbyl group can be eliminated, againforming the hydroxy group; if desired, the obtained compound isconverted into a peracylated derivative at the hydroxy functions.

The carboxy group of the neuraminic acid can be converted into theamide, and eventually the 2-hydroxy group in its hydrocarbyl derivativesin both sequences and acylate the free amino group with the desired acidand, if desired, peracylate the free hydroxy groups, or perform thisperacylation in every step of the processes, e.g., at the beginning.

The conversion of the carboxylic group of N-acyl derivatives ofneuraminic acid or of their 2-hydrocarbyl-glycosides to thecorresponding amide can be performed directly starting from the acid, orfrom its metal or organic base salt, or indirectly preparing first theester of the acid, an anhydride, or a halogenide of the acid, and thenconverting these compounds into the amide.

A preferred method consists of activating the carboxylic group and thenreacting the intermediate with the desired amine, utilizing methodsknown in peptide chemistry, avoiding methods utilizing acidic or basicconditions. If metal salts of the acid, like sodium, are used, it isconvenient to treat the salt with an ion exchange resin of the Dowextype or a similar resin. As an example, it is possible to use thecondensation method in presence of carbodiimides, e.g.,dicyclohexylcarbodiimide, benzylisopropylcarbodiimide orbenzylethylcarbodiimide, in the presence of 1-hydroxybenzo-triazol, orthe condensation in the presence of N,N'carbonyl-diimidazol. Startingfrom the aforesaid acidic derivatives, like esters or halogenides, e.g.,bromides or chlorides, the transformation into the amide is carried outby direct treatment with the desired amine at relatively lowtemperature, e.g., room temperature or -5° C. to 10° C., or highertemperatures, e.g., between 30° and 120° C. Ketones, aromatichydrocarbides, dimethylformamide, dimethylsulfoxide, dioxane ortetrahydrofuran can be used as solvents. The starting esters can bealiphatic esters, e.g., ethyl or methyl esters, or aromatic esters,e.g., phenols.

The 2-O-hydrocarbyl derivatives of the starting compounds or of thecompounds already possessing the amine function are prepared accordingto the conditions known for the acetylation of aldehydes or ketones, orfor the preparation of glycosides. The 2-hydrocarbyl groups of theglycosides can be transformed at the hydroxy group at every step byhydrolysis with acids under mild conditions.

If acylation of the amine group of neuraminic acid is performed at theend of the procedure, e.g., after amide or glycoside formation, knownacylation methods are used, e.g., treatment of the compound with acidhalogenides or anhydrides, eventually in the presence of inorganic ororganic bases, like pyridine or collidine. This acylation can beperformed contemporaneously with the acylation of hydroxy groups.

The transformation of the final compounds into their salts, as well asthe interconversion of the salts, is performed in a known manner, as forexample when intermediate salts are prepared for their purification.

The aforesaid procedure according to the present invention alsocomprises all variations in which the procedure is stopped at everystep, or in which the starting compound is an intermediate, or in whichthe starting compounds are prepared in situ.

The synthesis of the compounds of the present invention is illustratedby the following examples.

EXAMPLE 1 Butilamide of N-acetylneuraminic acid

3.23 g (10 mM) of N-acetylneuraminic acid methylester, preparedaccording to Kuhn et al., Chem Ber. 99,611 (1966), were solubilized in50 ml of anhydrous methyl alcohol; 3.66 g (50 mM) of 2-butylamine wereadded. The mixture was stirred for 5 hours at 40° C. The solution wasevaporated under vacuum and the residue was purified by silica gelchromatography, using as solvent a mixture of methylene chloride/methylalcohol/water, 110:40:6. The fractions containing the butilamide ofN-acetyl-neuraminic acid were gathered and evaporated under vacuum. Theresidue was crystallized from 50 ml of n-propyl alcohol. Yield: 85%.

Rf=0.25, chloroform/methyl alcohol/water, 110:40:6.

EXAMPLE 2 β-2-O-ethylglycoside of butilamide of N-acetylneuraminic acid

3.65 g (10 mM) of the β-2-O-ethylglycoside of N-acetylneuraminic acidethyl ester, prepared according to Kuhn et al., Chem Ber. 99, 611(1966), were solubilized in 80 ml of anhydrous methyl alcohol; 3.66 g(50 mM) of 2-butylamine were added. The mixture was stirred for 5 hoursat 40° C. The solution was evaporated under vacuum and the residue waspurified by silica gel chromatography, using as solvent a mixture ofmethylene chloride/methyl alcohol/water, 80:20:2. The fractionscontaining the β-2-O-ethylglycoside of the butilamide ofN-acetyl-neuraminic acid were gathered and evaporated under vacuum. Theresidue was crystallized from 50 ml of n-propyl alcohol and 100 ml ofethyl ether. Yield: 70%.

Rf=0.37, chloroform/methyl alcohol/water, 10:40:6;

0.19, chloroform/methyl alcohol/2.5N NH₄ OH, 0:20:2.

EXAMPLE 3 β-2-O-ethylglycoside of the benzylamide of N-acetylneuraminicacid

3.65 g (10 mM) of the β-2-O-ethylglycoside of N-acetylneuraminic acidethyl ester, prepared according to Kuhn et al., Chem Ber. 99, 611(1966), were solubilized in 50 ml of anhydrous methyl alcohol; 5.36 g(50 mM) of benzylamine were added. The mixture was stirred for 5 hoursat 40° C. The solution was evaporated under vacuum and the residue waspurified by silica gel chromatography, using as solvent a mixture ofmethylene chloride/methyl alcohol/water, 80:20:2. The fractionscontaining the β-2-O-ethylglycoside of the benzylamide ofN-acetyl-neuraminic acid were gathered and evaporated under vacuum. Theresidue was crystallized from 50 ml of isopropyl alcohol. Yield: 65%.

Rf=0.50, chloroform/methyl alcohol/water, 110:40:6;

0.16, chloroform/methyl alcohol/2.5N NH₄ OH, 80:20:2.

EXAMPLE 4 β-2-O-ethylglycoside of the dimethylaminopropylamide ofN-acetylneuraminic acid

3.65 g (10 mM) of the β-2-O-ethylglycoside of N-acetylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 10.2g (100 mM) of dimethylaminopropylamine were added. The mixture wasstirred overnight at 25 ° C. The solution was evaporated under vacuumand the residue was purified by silica gel chromatography, using assolvent a mixture of methylene chloride/methyl alcohol/2.5N NH₄ OH,55:45:10. The fractions containing the β-2-O-ethylglycoside ofdimethylaminopropylamide of N-acetyl-neuraminic acid were gathered andevaporated under vacuum. The residue was dissolved in 50 ml of water.Yield: 75%.

Rf=0.19, chloroform/methyl alcohol/2.5N NH₄ OH, 40:60:15.

EXAMPLE 5 β-2-O-ethylglycoside of the dimethylaminopropylamide ofN-acetylneuraminic acid (maleic acid salt)

3.65 g (10 mM) of the β-2-O-ethylglycoside of N-acetylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 10.2g (100 mM) of dimethylaminopropylamine were added. The mixture wasstirred overnight at 25° C. The solution was evaporated under vacuum andthe residue was purified by silica gel chromatography, using as solventa mixture of methylene chloride/methyl alcohol/2.5N NH₄ OH, 55:45:10.The fractions containing the β-2-O-ethylglycoside of thedimethylaminopropylamide of N-acetyl-neuraminic acid were gathered andevaporated under vacuum. The residue was dissolved in 50 ml of water, astoichiometric amount of maleic acid was added, and the material waslyophilized. Yield: 75%.

Rf=0.19, chloroform/methyl alcohol/2.5N NH₄ OH, 40:60:15.

EXAMPLE 6 β-2-O-ethylglycoside of the dimethylamide ofN-acetylneuraminic acid

3.65 g (10 mM) of the β-2-O-ethylglycoside of N-acetylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 4.5 g(100 mM) of dimethylamine were added. The mixture was stirred overnightat 25° C. The solution was evaporated under vacuum and the residue waspurified by silica gel chromatography, using as solvent a mixture ofmethylene chloride/methyl alcohol/water, 80:20:2. The fractionscontaining the β-2-O-ethylglycoside of the dimethylamide ofN-acetyl-neuraminic acid were gathered and evaporated under vacuum. Theresidue was crystallized from 30 ml of methanol and 150 ml of ethylether. Yield: 80%.

Rf=0.38, chloroform/methyl alcohol/water, 110:40:6.

EXAMPLE 7 β-2-O-ethylglycoside of the dimethylaminopropylamide ofN-palmitoylneuraminic acid

5.62 g (10 mM) of the β-2-O-ethylglycoside of N-palmitoylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 10.2g (100mM) of dimethylaminopropylamine were added. The mixture wasstirred overnight at 25° C. The solution was evaporated under vacuum andthe residue was purified by silica gel chromatography, using as solventa mixture of methylene chloride/methyl alcohol/water, 110:40:6. Thefractions containing the β-2-O-ethylglycoside of thedimethylaminopropylamide of N-palmitoyl-neuraminic acid were gatheredand evaporated under vacuum. The residue was dissolved in 60 ml of waterand lyophilized. Yield: 70%.

Rf=0.12, chloroform/methyl alcohol/2.5N NH₄ OH, 80:20:2.

EXAMPLE 8 β-2-O-ethylglycoside of the dimethylaminopropylamide ofN-palmitoylneuraminic acid (maleic acid salt)

5.62 g (10 mM) of the β-2-O-ethylglycoside of N-palmitoylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 10.2g (100 mM) of dimethylaminopropylamine were added. The mixture wasstirred overnight at 25° C. The solution was evaporated under vacuum andthe residue was purified by silica gel chromatography, using as solventta mixture of methylene chloride/methyl alcohol/water, 110:40:6. Thefractions containing the β-2-O-ethylglycoside of thedimethylaminopropylamide of N-palmitoyl-neuraminic acid were gatheredand evaporated under vacuum. The residue was dissolved in 50 ml ofwater, a stoichiometric amount of maleic acid was added, and thematerial was lyophilized. Yield: 70%.

Rf=0.12, chloroform/methyl alcohol/2.5N NH₄ OH, 0:60:15.

EXAMPLE 9 α-2-O-ethylglycoside of the dimethylaminopropylamide ofN-palmitoylneuraminic acid

5.48 g (10 mM) of the α-2-O-ethylglycoside of N-palmitoylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 10.2g (100 mM) of dimethylaminopropylamine were added. The mixture wasstirred overnight at 25° C. The solution was evaporated under vacuum andthe residue was purified by silica gel chromatography, using as solventa mixture of methylene chloride/methyl alcohol/water, 110:40:6. Thefractions containing the α-2-O-ethylglycoside of thedimethylaminopropylamide of N-palmitoyl-neuraminic acid were gatheredand evaporated under vacuum. The residue was dissolved in 60 ml of waterand lyophilized. Yield: 70%.

Rf=0.40, chloroform/methyl alcohol/0.3% CaCl₂, 60:40:9.

EXAMPLE 10 α-2-O-ethylglycoside of the dimethylaminopropylamide ofN-palmitoylneuraminic acid (maleic acid salt)

5.48 g (10 mM) of the α-2-O-ethylglycoside of N-palmitoylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 10.2g (100 mM) of dimethylaminopropylamine were added. The mixture wasstirred overnight at 25° C. The solution was evaporated under vacuum andthe residue was purified by silica gel chromatography, using as solventa mixture of methylene chloride/methyl alcohol/water, 110:40:6. Thefractions containing the α-2-O-ethylglycoside of thedimethylaminopropylamide of N-palmitoyl-neuraminic acid were gatheredand evaporated under vacuum. The residue was dissolved in 50 ml ofwater, a stoichiometric amount of maleic acid was added, and thematerial was lyophilized. Yield: 70%.

Rf=0.40, chloroform/methyl alcohol/0.3% CaCl₂, 60:40:9.

EXAMPLE 11 β-2-O-ethylglycoside of the dimethylamide ofN-palmitoylneuraminic acid

5.56 g (10 mM) of the α-2-O-ethylglycoside of N-palmitoyl-neuraminicacid, sodium salt, were dissolved in 50 ml of pyridine, and 2.3 g (20mM) of pyridinium chloride and 4.12 g (20 mM) ofN,N'dicyclohexyl-carbodiimide were added. The mixture was stirred for 2hours at 25° C. 4.5 g (100 mM) of dimethylamine were added and thereaction was conducted overnight at 25° C. The solution was evaporatedunder vacuum and the residue was purified by silica gel chromatography,using as solvent a mixture of methylene chloride/methyl alcohol/water,80:10:1. The fractions containing the β-2-O-ethylglycoside of thedimethylamide of N-palmitoyl-neuraminic acid were gathered andevaporated under vacuum. The residue was dissolved in 50 ml of acetoneand precipitated in 20 volumes of hexane. Yield: 90%.

Rf=0.69, chloroform/methyl alcohol/water, 110:40:6.

EXAMPLE 12 α-2-O-ethylglycoside of the dimethylamide ofN-palmitoylneuraminic acid

5.48 g (10 mM) of the α-2-O-ethylglycoside of N-palmitoylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 4.5 g(100 mM) of dimethylamine were added. The mixture was stirred overnightat 25° C. The solution was evaporated under vacuum and the residue waspurified by silica gel chromatography, using as solvent a mixture ofmethylene chloride/methyl alcohol/water, 80:10:1. The fractionscontaining the α-2-O-ethylglycoside of the dimethylamide ofN-palmitoyl-neuraminic acid were gathered and evaporated under vacuum.The residue was dissolved in 50 ml of acetone and precipitated in 20volumes of hexane. Yield: 90%.

Rf=0.69, chloroform/methyl alcohol/water, 110:40:6.

EXAMPLE 13 α-2-O-ethylglycoside of the butylamide of N-acetylneuraminicacid

3.65 g (10 mM) of the α-2-O-ethylglycoside of N-acetylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 3.66g (50 mM) of butylamine were added. The mixture was stirred overnight at25° C. The solution was evaporated under vacuum and the residue waspurified by silica gel chromatography, using as solvent a mixture ofmethylene chloride/methyl alcohol/water, 80:20:2. The fractionscontaining the α-2-O-ethylglycoside of the butylamide ofN-acetyl-neuraminic acid were gathered and evaporated under vacuum. Theresidue was crystallized from 50 ml of methanol and 300 ml of ethylether. Yield: 75%.

Rf=0.55, chloroform/methyl alcohol/water, 110:40:6;

Rf=0.53, chloroform/methyl alcohol/2.5N NH₄ OH, 40:60:15.

EXAMPLE 14 α-2-O-ethylglycoside of the dimethylaminopropylamide ofN-acetylneuraminic acid

3.65 g (10 mM) of the α-2-O-ethylglycoside of N-acetylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 10.2g (100 mM) of dimethylaminopropylamine were added. The mixture wasstirred overnight at 25° C. The solution was evaporated under vacuum andthe residue was purified by silica gel chromatography, using as solventa mixture of methylene chloride/methyl alcohol/2.5N NH₄ OH, 55:45:10.The fractions containing the α-2-O-ethylglycoside of thedimethylaminopropylamide of N-acetyl-neuraminic acid were gathered andevaporated under vacuum. The residue was dissolved in 50 ml of water andlyophilized. Yield: 70%.

Rf=0.21, chloroform/methyl alcohol/2.5N NH₄ OH, 40:60:15.

EXAMPLE 15 α-2-O-ethylglycoside of the dimethylaminopropylamide ofN-acetylneuraminic acid (maleic acid salt)

3.65 g (10 mM) of the β-2-O-ethylglycoside of N-acetylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 10.2g (100 mM) of dimethylaminopropylamine were added. The mixture wasstirred overnight at 25° C. The solution was evaporated under vacuum andthe residue was purified by silica gel chromatography, using as solventa mixture of methylene chloride/methyl alcohol/2.5N NH₄ OH, 55:45:10.The fractions containing the α-2-O-ethylglycoside of thedimethylaminopropylamide of N-acetyl-neuraminic acid were gathered andevaporated under vacuum. The residue was dissolved in 50 ml of water, astoichiometric amount of maleic acid was added, and the material waslyophilized. Yield: 70%.

Rf=0.21, chloroform/methyl alcohol/2.5N NH₄ OH, 40:60:15.

EXAMPLE 16 α-2-O-ethylglycoside of N-acetylneuraminic acid amide withL-alanine-D-isoglutamine

3.65 g (10 mM) of the α-2-O-ethylglycoside of N-acetylneuraminic acidethyl ester were solubilized in 40 ml of water, and 10 ml (10 mM) ofNaOH were added. The solution was maintained at 25° C. for 30 minutes,neutralized with 1N HCl, and eluted with water from a column containing30 ml of Dowex 50x8 resin, pyridinium form. The eluate was lyophilizedand the residue was solubilized in 100 ml of anhydrous pyridine. 1.15 g(10 mM) of N-hydroxysuccinimide and 4.13 g (20 mM) ofN,N'-dicyclohexylcarbodiimide were added at -10° C. After 15 minutes,the temperature was raised to 25° C., and the mixture was stirred for 5hours. 5.14 g (15 mM) of L-alanine-D-isoglutamine benzyl esterhydrochloride (prepared according to Le Francier and Kusumoto) wereadded at 25° C. The mixture was stirred overnight and then evaporatedunder vacuum. The obtained residue was dissolved in 200 ml of a mixtureof n-butanol/water/acetic acid, 4:1:1, and hydrogenated in an H₂ currentin the presence of BaSO₄ -supported palladium. After filtration, thesolution was evaporated, and the residue was purified by silica gelchromatography, using as solvent a mixture of methylene chloride/methylalcohol/water, 60:35:8. The fractions containing theα-2-O-ethylglycoside of N-acetyl-neuraminic acid amide withn-alanine-D-isoglutamine were gathered and evaporated under vacuum. Theresidue was dissolved in 200 ml of water and lyophilized. Yield: 60%.

Rf=0.56, chloroform/methyl alcohol/2.5N NH₄ OH, 60:35:8;

Rf=0.18, chloroform/methyl alcohol/0.3% CaCl₂, 60:40:9.

EXAMPLE 17 Peracetylated β-2-O-ethylglycoside of N-palmitoyl-neuraminicacid amide with L-alanine-D-isoglutamine

5.56 g (10 mM) of the α-2-O-ethylglycoside of N-palmitoylneuraminicacid, sodium salt, were solubilized in 50 ml of anhydrousN,N'-dimethylformamide at 25° C.; 3.06 g (11 mM) of p-bromophenacylbromide were added and the solution was stirred overnight. 18 ml ofanhydrous pyridine and 10.2 g of acetic anhydride were added andstirring was conducted for 24 hours at 35° C. The solution wasevaporated under vacuum and the residue was dissolved with 100 ml ofwater and extracted three times with 200 ml of methylene chloride. Theorganic phases were washed twice with 50 ml of water and then gathered,anhydrified with anhydrous sodium sulfate, and evaporated under vacuum.The obtained residue was solubilized in 50 ml of anhydrousN,N'-dimethylformamide at 25° C.; 2.64 g (20 mM) of sodium thiophenatewere added and the mixture was stirred for 4 hours. The solution wasevaporated under high vacuum. The residue was extracted three times with200 ml of ethyl acetate, washed with 100 ml of cold 1N HCl and twicewith 50 ml of water. The organic phases were anhydrified with anhydroussodium sulfate, gathered, and evaporated under vacuum. The residue wasdissolved in 30 ml of water and eluted with water from a columncontaining 30 ml of Dowex 50X8 resin, pyridinium form. The eluate waslyophilized and the residue was solubilized in 100 ml of anhydrouspyridine. 1.15 g (10 mM) of N-hydroxysuccinimide and 4.13 g (20 mM) ofN,N'-dicyclohexylcarbodiimide were added at -10° C. After 15 minutes thetemperature was raised to 25° C. and the mixture was stirred for 5hours. 5.14 g (15 mM) of L-alanine-D-isoglutamine benzyl esterhydrochloride (prepared according to Le Francier and Kusumoto) wereadded at 25° C. The mixture was stirred overnight and then evaporatedunder vacuum. The obtained residue was dissolved in 200 ml of a mixtureof N-butanol/water/acetic acid, 4:1:1, and hydrogenated in an H₂ currentin the presence of BaSO₄ -supported palladium. After filtration, thesolution was evaporated, and the residue was purified by silica gelchromatography, using as solvent a mixture of methylene chloride/methylalcohol/water, 60:35:8. The fractions containing theα-2-O-ethylglycoside of N-acetyl-neuraminic acid amide withL-alanine-D-isoglutamine were gathered and evaporated under vacuum. Theresidue was dissolved in 200 ml of water and lyophilized. Yield: 55%.

Rf=0.54, chloroform/methanol, 90:10.

EXAMPLE 18 β-2-O-ethlglycoside of N-acetylneuraminic acid amide withL-alanine-D-isoglutamine

3.59 g (10 mM) of the β-2-O-ethylglycoside of N-acetylneuraminic acid,sodium salt, prepared according to Eschenfelder and Brossmer, HoppeSeyler's Z. Physiol. Chem. 360, 1253 (1979), were solubilized in 50 mlof anhydrous N,N'-dimethylformamide at 25° C.; 3.06 g (11 mM) ofp-bromophenacyl bromide were added and the solution was stirredovernight. 18 ml of anhydrous pyridine and 10.2 g of acetic anhydridewere added and stirring was conducted for 24 hours at 35° C. Thesolution was evaporated under vacuum and the residue was dissolved with100 ml of water and extracted three times with 200 ml of methylenechloride. The organic phases were washed twice with 50 ml of water andthen gathered, anhydrified with anhydrous sodium sulfate, and evaporatedunder vacuum. The obtained residue was solubilized in 50 ml of anhydrousN,N'-dimethylformamide at 25° C.; 2.64 g (20 mM) of sodium thiophenatewere added aid the mixture was stirred for 4 hours. The solution wasevaporated under high vacuum. The residue was extracted three times with200 ml of ethyl acetate, washed with 100 ml of cold 1N HCl and twicewith 50 ml of water. The organic phases were anhydrified with anhydroussodium sulfate, gathered, and evaporated under vacuum. The residue wasdissolved in 30 ml of water and eluted with water from a columncontaining 30 ml of Dowex 50x8 resin, pyridinium form. The eluate waslyophilized and the residue was solubilized in 100 ml of anhydrouspyridine. 1.15 g (10 mM) of N-hydroxysuccinimide and 4.13 g (20 mM) ofN,N'-dicyclohexylcarbodiimide were added at -10° C. After 15 minutes,the temperature was raised to 25° C. and the mixture was stirred for 5hours. 5.14 g (15 mM) of L-alanine-D-isoglutamine benzyl esterhydrochloride (prepared according to Le Francier and Kusumoto) wereadded at 25° C. The mixture was stirred overnight and then evaporatedunder vacuum. The obtained residue was dissolved in 60 ml of anhydrousmethanol at 25° C. 100 mg of potassium terbutylate were added, and themixture was stirred for 30 minutes. 5 ml of anhydrous Dowex 50×8 resin,H⁺ form, were added. The solution was filtered and evaporated undervacuum, the residue was dissolved in 30 ml of water, and 10 ml of 1NNaOH were added. The solution was stirred for 15 minutes at 25° C. andeluted with water from a column containing 30 ml of Dowex 50x8 resin, H⁺form. The eluate was lyophilized, and purified by silica gelchromatography, using as solvent a mixture of methylene chloride/methylalcohol/water, 60:40:9. The fractions containing theβ-2-O-ethylglycoside of N-acetyl-neuraminic acid amide withL-alanine-D-isoglutamine were gathered and evaporated under vacuum. Theresidue was dissolved in 50 ml of water and lyophilized. Yield: 60%

Rf=0.12, chloroform/methyl alcohol/2.5N NH₄ OH, 60:35:8;

0.10, chloroform/methyl alcohol/0.3% CaCl₂, 60:40:9.

EXAMPLE 19 β-2-O-ethylglycoside of N-palmitoylneuraminic acid amide withL-alanine-D-isoglutamine

5.56 g (10 mM) of the β-2-O-ethylglycoside of N-palmitoylneuraminicacid, sodium salt, were solubilized in 50 ml of anhydrousN,N'-dimethylformamide at 25° C.; 3.06 g (11 mM) of p-bromophenacylbromide were added and the solution was stirred overnight. 18 ml ofanhydrous pyridine and 10.2 g of acetic anhydride were added andstirring was conducted for 24 hours at 35° C. The solution wasevaporated under vacuum and the residue was dissolved with 100 ml ofwater and extracted three times with 200 ml of methylene chloride. Theorganic phases were washed twice with 50 ml of water and then gathered,anhydrified with anhydrous sodium sulfate and evaporated under vacuum.The obtained residue was solubilized in 50 ml of anhydrousN,N'-dimethylformamide at 25° C.; 2.64 g (20 mM) of sodium thiophenatewere added and the mixture was stirred for 4 hours. The solution wasevaporated under high vacuum. The residue was extracted three times with200 ml of ethyl acetate, washed with 100 ml of cold 1N HCl and twicewith 50 ml of water. The organic phases were anhydrified with anhydroussodium sulfate, gathered, and evaporated under vacuum. The residue wasdissolved in 30 ml of water and eluted with water from a columncontaining 30 ml of Dowex 50x8 resin, pyridinium form. The eluate waslyophilized and the residue was solubilized in 100 ml of anhydrouspyridine. 1.15 g (10 mM) of N-hydroxysuccinimide and 4.13 g (20 mM) ofN,N'-dicyclohexylcarbodiimide were added at -10° C. After 15 minutes,the temperature was raised to 25° C. and the mixture was stirred for 5hours. 5.14 g (15 mM) of L-alanine-D-isoglutamine benzyl esterhydrochloride (prepared according to Le Francier and Kusumoto) wereadded at 25° C. The mixture was stirred overnight and then evaporatedunder vacuum. The obtained residue was dissolved in 60 ml of anhydrousmethanol at 25° C., 100 mg of potassium terbutylate were added, and themixture was stirred for 30 minutes. 5 ml of anhydrous Dowex 50x8 resin,H⁺ form, were added. The solution was filtered and evaporated undervacuum. The residue was dissolved in 30 ml of water, and 10 ml of 1NNaOH were added. The solution was stirred for 15 minutes at 25° C. andeluted with water from a column containing 30 ml of Dowex 50x8 resin, H⁺form. The eluate was lyophilized, and purified by silica gelchromatography, using as solvent a mixture of methylene chloride/methylalcohol/water, 60:40:9. The fractions containing theβ-2-O-ethylglycoside of N-palmitoyl-neuraminic acid amide withL-alanine-D-isoglutamine were gathered and evaporated under vacuum. Theresidue was dissolved in 50 ml of a mixture of water/dioxane, 4:1, andlyophilized. Yield: 60%.

Rf=0.12, chloroform/methyl alcohol/2.5N NH₄ OH, 110:40:6.

0.57, chloroform/methyl alcohol/0.3% CaCl₂, 60:40:9.

EXAMPLE 20 α-2-O-ethylglycoside of N-acetylneuraminic acid amide witharginine

3.65 g (10 mM) of the α-2-O-ethylglycoside of N-acetylneuraminic acidethyl ester, prepared according to van der Vlengel et al., Carbohydr.Res. 102, 121 (1982), were solubilized in 40 ml of water and 10 ml (10mM) of NaOH were added. The solution was maintained at 25° C. for 30minutes, neutralized with 1N HCl, and eluted with water from a columncontaining 30 ml of Dowex 50x8 resin, pyridinium form. The eluate waslyophilized and the residue was solubilized in 100 ml of anhydrouspyridine. 1.15 g (10 mM) of N-hydroxysuccinimide and 4.13 g (20 mM) ofN,N'-dicyclohexylcarbodiimide were added at -10° C. After 15 minutes,the temperature was raised to 25° C. and the mixture was stirred for 5hours. 4.64 g (15mM) of N-nitro-L-arginine benzyl ester (preparedaccording to Bonnaud, Bull. Chim. Farm. 121, 1982) were added at 25° C.The mixture was stirred overnight and then evaporated under vacuum. Theobtained residue was dissolved in 200 ml of a mixture ofn-butanol/water/acetic acid, 4:1:1, and hydrogenated in an H₂ current inthe presence of BaSO₄ -supported palladium. After filtration, thesolution was evaporated, and the residue was purified by silica gelchromatography, using as solvent a mixture of methylene chloride/methylalcohol/water, 60:40:9. The fractions containing theα-2-O-ethylglycoside of N-acetyl-neuraminic acid amide with argininewere gathered and evaporated under vacuum. The residue was dissolved in50 ml of water and lyophilized. Yield: 50%.

Rf=0.13, chloroform/methyl alcohol/0.3% CaCl₂, 60:40:9.

EXAMPLE 21 β-2-O-ethylglycoside of N-acetylneuraminic acid amide witharginine

3.59 g (10 mM) of the β-2-O-ethylglycoside of N-acetylneuraminic acid,sodium salt, were solubilized in 50 ml of anhydrousN,N'-dimethylformamide at 25° C.; 3.06 g (11 mM) of p-bromophenacylbromide were added and the solution was stirred overnight. 18 ml ofanhydrous pyridine and 10.2 g of acetic anhydride were added andstirring was conducted for 24 hours at 35° C. The solution wasevaporated under vacuum and the residue was dissolved with 100 ml ofwater and extracted three times with 200 ml of methylene chloride. Theorganic phases were washed twice with 50 ml of water and then gathered,anhydrified with anhydrous sodium sulfate, and evaporated under vacuum.The obtained residue was solubilized in 50 ml of anhydrousN,N'-dimethylformamide at 25° C.; 2.64 g (20 mM) of sodium thiophenatewere added and the mixture was stirred for 4 hours. The solution wasevaporated under high vacuum. The residue was extracted three times with200 ml of ethyl acetate, washed with 100 ml of cold 1N HCl and twicewith 50 ml of water. The organic phases were anhydrified with anhydroussodium sulfate, gathered, and evaporated under vacuum. The residue wassolubilized in 100 ml of anhydrous pyridine; 1.15 g (10 mM) ofN-hydroxysuccinimide, 4.13 g (20 mM) of N,N'-dicyclohexylcarbodiimideand 11.6 g (10 mM) of pyridinium chloride were added at -10° C. After 15minutes, the temperature was raised to 25° C. and the mixture wasstirred for 5 hours. 4.64 g (15 mM) of N-nitro-L-arginine benzyl esterhydrochloride (prepared according to Bonnaud, Bull. Chim. Farm. 121,1982) were added at 25° C. The mixture was stirred overnight and thenevaporated under vacuum. The obtained residue was dissolved in 60 ml ofanhydrous methanol at 25° C. 100 mg of potassium terbutylate were added,and the mixture was stirred for 30 minutes. 5 ml of anhydrous Dowex 50x8resin, H⁺ form, were added. The solution was filtered and evaporatedunder vacuum, and the residue was dissolved in 100 ml of a mixture ofN-butanol/water/acetic acid, 4:1:1, and hydrogenated in an H₂ current inthe presence of BaSO₄ -supported palladium. After filtration, thesolution was evaporated, and the residue was purified by silica gelchromatography, using as solvent a mixture of methylene chloride/methylalcohol/water, 110:40:6. The fractions containing theβ-2-O-ethylglycoside of N-palmitoyl-neuraminic acid amide with argininewere gathered and evaporated under vacuum. The residue was dissolved in50 ml of water and lyophilized. Yield: 50%.

Rf=0.10, chloroform/methyl alcohol/0.3% CaCl₂, 60:40:9.

EXAMPLE 22 β-2-O-ethylglycoside of N-palmitoylneuraminic acid amide witharginine

5.56 g (10 mM) of the β-2-O-ethylglycoside of N-palmitoyl-neuraminicacid, sodium salt, were solubilized in 50 ml of anhydrousN,N'-dimethylformamide at 25° C.; 3.06 g (11 mM) of p-bromophenacylbromide were added and the solution was stirred overnight. 18 ml ofanhydrous pyridine and 10.2 g of acetic anhydride were added andstirring was conducted for 24 hours at 35° C. The solution wasevaporated under vacuum and the residue was dissolved with 100 ml ofwater and extracted three times with 200 ml of methylene chloride. Theorganic phases were washed twice with 50 ml of water and then gathered,anhydrified with anhydrous sodium sulfate, and evaporated under vacuum.The obtained residue was solubilized in 50 ml of anhydrousN,N'-dimethylformamide at 25° C.; 2.64 g (20 mM) of sodium thiophenatewere added and the mixture was stirred for 4 hours. The solution wasevaporated under high vacuum. The residue was extracted three times with200 ml of ethyl acetate, washed with 100 ml of cold 1N HCl and twicewith 50 ml of water. The organic phases were anhydrified with anhydroussodium sulfate, gathered, and evaporated under vacuum. The residue wasdissolved in 30 ml of water and eluted with water from a columncontaining 30 ml of a Dowex 50x8 Dowex resin, pyridinium form. Theeluate was lyophilized and then solubilized in 100 ml of anhydrouspyridine; 1.15 g (10 mM) of N-hydroxysuccinimide, 4.13 g (20 mM) ofN,N'-dicyclohexylcarbodiimide and 11.6 g (10 mM) of pyridinium chloridewere added at -10° C. After 15 minutes, the temperature was raised to25° C. and the mixture was stirred for 5 hours. 4.64 g (15 mM) ofN-nitro-L-arginine benzyl ester hydrochloride (prepared according toBonnaud, Bull. Chim. Farm. 121, 1982) were added at 25° C. The mixturewas stirred overnight and then evaporated under vacuum. The obtainedresidue was dissolved in 60 ml of anhydrous methanol at 25° C. 100 mg ofpotassium terbutylate were added, and the mixture was stirred for 30minutes. 5 ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. Thesolution was filtered and evaporated under vacuum. The residue wasdissolved in 100 ml of a mixture of n-butanol/water/acetic acid, 4:1:1,and hydrogenated in an H₂ current in the presence of BaSO₄ -supportedpalladium. After filtration, the solution was evaporated, and theresidue was purified by silica gel chromatography, using as solvent amixture of methylene chloride/methyl alcohol/1.5M acetic acid, 110:40:6.The fractions containing the β-2-O-ethylglycoside ofN-palmitoyl-neuraminic acid amide with arginine were gathered andevaporated under vacuum. The residue was crystallized from a mixture of100 ml of methanol and 300 ml of ethyl ether. Yield: 60%.

Rf=0.12, chloroform/methyl alcohol/H₂ O, 110:40:6.

EXAMPLE 23 α-2-O-ethylglycoside Of N-palmitoyl-neuraminic acid amidewith arginine

5.56 g (10 mM) of the α-2-O-ethylglycoside of N-palmitoyl-neuraminicacid, sodium salt, were solubilized in 40 ml of water and eluted withwater from a column containing 30 ml of 50x8 Dowex resin, pyridiniumform. The eluate was lyophilized and then solubilized in 100 ml ofanhydrous pyridine. 1.15 g (10 mM) of N-hydroxysuccinimide, 4.13 g (20mM) of N,N'-dicyclohexylcarbodiimide, and 11.6 g (10 mM) of pyridiniumchloride were added at -10° C. After 15 minutes, the temperature wasraised to 25° C. and the mixture was stirred for 5 hours. 4.64 g (15 mM)of N-nitro-L-arginine benzyl ester hydrochloride (prepared according toBonnaud, Bull. Chim. Farm. 121, 1982) were added at 25° C. The mixturewas stirred overnight and then evaporated under vacuum. The residue wasdissolved in 100 ml of a mixture of n-butanol/water/acetic acid, 4:1:1,and hydrogenated in an H₂ current in the presence of BaSO₄ -supportedpalladium. After filtration, the solution was evaporated, and theresidue was purified by silica gel chromatography, using as solvent amixture of methylene chloride/methyl alcohol/1.5M acetic acid, 110:40:6.The fractions containing the α-2-O-ethylglycoside ofN-palmitoyl-neuraminic acid amide with arginine were gathered andevaporated under vacuum. The residue was crystallized from a mixture of40 ml of methanol and 150 ml of ethyl ether. Yield: 70%.

Rf=0.14, chloroform/methyl alcohol/H₂ O, 110:40:6;

0.32, chloroform/methyl alcohol/2.5N NH₄ OH, 60:35:8.

EXAMPLE 24 Dimethylamide of N-palmitoylneuraminic acid

5.34 g (10 mM) of the α-2-O-ethylglycoside of N-palmitoyl-neuraminicacid were suspended in a mixture of 100 ml of 0.1M H₂ SO₄ /ethanol, 4:1,at 60° C., and stirred for 16 hours. The product was extracted once with100 ml of ethyl acetate. The organic phases were washed three times with50 ml of water, gathered, and evaporated under vacuum. The obtainedresidue was dissolved in 200 ml of anhydrous methanol at 25° C. 20 ml ofanhydrous Dowex 50x8 resin, H⁺ form, were added. The mixture was stirredfor 2 hours. To the filtered solution, 4.5 g (100 mM) of dimethylaminewere added at 25° C. and the solution was stirred for 24 hours. Themixture was evaporated, and the residue was purified by silica gelchromatography, using as solvent a mixture of methylene chloride/methylalcohol/water, 80:20:2. The fractions containing the dimethylamide ofN-acetyl-neuraminic acid were gathered and evaporated under vacuum. Theresidue was dissolved in 60 ml of a mixture of water/dioxane, 4:1, andlyophilized. Yield: 75%.

Rf=0.63, chloroform/methyl alcohol/H₂ O, 110:40:6.

EXAMPLE 25 β-2-ethylglycoside morpholino-propylamide ofN-palmitoylneuraminic acid

5.62 g (10 mM) of β-2-ethylglycoside N-palmitoylneuraminic acid ethylester were solubilized in 50 ml of anhydrous methanol; 14.4 g (100 mM)of N-(3-aminopropyl)-morpholine were added. The mixture was stirredovernight at 25° C. The solution was evaporated under vacuum and theresidue was purified by silica gel chromatography, using as solvent amixture of methylene chloride/methyl alcohol/water, 80:10:1. Thefractions containing the compound were gathered and evaporated undervacuum. The residue was dissolved in 100 ml of water and lyophilized.Yield: 85%.

Rf=0.30, chloroform/methyl alcohol/H₂ O, 80:20:2;

0.62, chloroform/methyl alcohol/2.5N NH₄ OH, 110:40:6.

EXAMPLE 26 β-2-ethylglycoside morpholino-propylamide ofN-palmitoylneuraminic acid (maleic acid salt)

5.56 g (10 mM) of β-2-O-ethylglycoside N-palmitoylneuraminic acid ethylester were solubilized in 50 ml of pyridine; 2.3 g (20 mM) of pyridiniumchloride and 4.12 g (20 mM) of N,N'-dicyclohexylcarbodiimide were added.The mixture was stirred for 2 hours at 25° C. 14.4 g (100 mM) ofN-(3-aminopropyl)-morpholine were added. The mixture was stirredovernight at 25° C. The solution was evaporated under vacuum and theresidue was purified by silica gel chromatography, using as solvent amixture of methylene chloride/methyl alcohol/water, 80:10:1. Thefractions containing the β-2-ethylglycoside morpholino-propylamide ofN-palmitoylneuraminic acid were gathered and evaporated under vacuum.The residue was dissolved in 100 ml of water. After adding astoichiometric amount of maleic acid, the mixture was lyophilized.Yield: 85%.

Rf=0.30, chloroform/methyl alcohol/H₂ O, 80:20:2;

0.62, chloroform/methyl alcohol/2.5N NH₄ OH, 110:40:6.

EXAMPLE 27 Dimethylaminopropylamide of N-palmitoyl-neuraminic acid

5.34 g (10 mM) of the α-2-O-ethylglycoside of N-palmitoyl-neuraminicacid were suspended in a mixture of 100 ml of 0.1M H₂ SO₄ /ethanol, 4:1,at 60° C., and stirred for 16 hours. The product was extracted once with200 ml of ethyl acetate and then twice with 100 ml of ethyl acetate; theorganic phases were washed three times with 50 ml of water, gathered,and evaporated under vacuum. The obtained residue was dissolved in 200ml of anhydrous methanol at 25° C. 20 ml of anhydrous Dowex 50x8 resin,H⁺ form, were added. The mixture was stirred for 2 hours. To thefiltered solution 10.2 g (100 mM) of dimethyl-aminopropylamine wereadded at 25 C. and the solution was stirred for 24 hours. The mixturewas evaporated, and the residue was purified by silica gelchromatography, using as solvent a mixture of methylene chloride/methylalcohol/2.5N NH₄ OH, 60:35:8. The fractions containing the compound weregathered and evaporated under vacuum. The residue was dissolved in 60 mlof a mixture of water/dioxane, 1:2, and lyophilized. Yield: 70%.

Rf=0.21, chloroform/methyl alcohol/0.3% CaCl₂, 60:40:9.

EXAMPLE 28 Dimethylaminopropylamide of N-palmitoyl-neuraminic acid(maleic acid salt)

5.34 g (10 mM) of the α-2-O-ethylglycoside of N-palmitoyl-neuraminicacid were suspended in a mixture of 100 ml of 0.1M H₂ SO₄ /ethanol, 4:1,at 60° C., and stirred for 16 hours. The product was extracted once with200 ml of ethyl acetate and then twice with 100 ml of ethyl acetate; theorganic phases were washed three times with 50 ml of water, gathered,and evaporated under vacuum. The obtained residue was dissolved in 200ml of anhydrous methanol at 25° C. 20 ml of anhydrous Dowex 50x8 resin,H⁺ form, were added. The mixture was stirred for 2 hours. To thefiltered solution 10.2 g (100 mM) of dimethyl-aminopropylamine wereadded at 25° C., and the solution was stirred for 24 hours. The mixturewas evaporated, and the residue was purified by silica gelchromatography, using as solvent a mixture of methylene chloride/methylalcohol/2.5N NH₄ OH, 60:35:8. The fractions containing the compound weregathered and evaporated under vacuum. The residue was dissolved in 60 mlof a mixture of water/dioxane, 1:2. A stoichiometric amount of maleicacid was added, and the mixture was lyophilized. Yield: 70%.

Rf=0.21, chloroform/methyl alcohol/0.3% CaCl₂, 60:40:9.

EXAMPLE 29 β-2-O-ethylglycoside butylamide of N-palmitoylneuraminic acid

5.62 g (10 mM) of β-2-O-ethylglycoside N-palmitoylneuraminic acid ethylester were solubilized in 50 ml of anhydrous methanol; 3.66 g (50 mM) of2-butylamine were added. The mixture was stirred for 5 hours at 40° C.The solution was evaporated under vacuum and the residue was purified bysilica gel chromatography, using as solvent a mixture of methylenechloride/methyl alcohol/water, 80:10:1. The fractions containing theβ-2-O-ethylglycoside butylamide of N-palmitoylneuraminic acid weregathered and evaporated under vacuum. The residue was dissolved in 60 mlof dioxane and lyophilized. Yield: 70%.

Rf=0.71, chloroform/methyl alcohol, 80:20;

0.60, chloroform/methyl alcohol/2.5N NH₄ OH, 80:20:2.

EXAMPLE 30 β-2-O-ethylglycoside dimethylaminoethylamide ofN-palmitoylneuraminic acid

5.62 g (10 mM) of β-2-O-ethylglycoside N-palmitoylneuraminic acid ethylester were solubilized in 50 ml of anhydrous methanol; 4.4 g (50 mM) of2-dimethylaminoethylamine were added. The mixture was stirred overnightat 40° C. The solution was evaporated under vacuum and the residue waspurified by silica gel chromatography, using as solvent a mixture ofmethylene chloride/methyl alcohol/2.5N NH₄ OH, 80:20:2. The fractionscontaining the β-2-O-ethylglycoside dimethylaminoethylamide ofN-palmitoylneuraminic acid were gathered and evaporated under vacuum.The residue was dissolved in 60 ml of water and lyophilized. Yield: 80%.

Rf=0.11, chloroform/methyl alcohol, 70:30;

0.43, chloroform/methyl alcohol/2.5N NH₄ OH, 110:40:6.

EXAMPLE 31 β-2-O-ethlglycoside dimethylaminoethylamide ofN-palmitoylneuraminic acid (maleic acid salt)

5.56 g (10 mM) of β-2-O-ethylglycoside N-palmitoylneuraminic acid weresolubilized in 50 ml of pyridine; 2.3 g (20 mM) of pyridinium chlorideand 4.12 g (20 mM) of N,N'-dicyclo-hexylcarbodiimide were added. Themixture was stirred for 2 hours at 25° C. 8.8 g (100 mM) ofdimethylaminoethylamine were added and the reaction was conductedovernight at 25° C. The solution was evaporated under vacuum and theresidue was purified by silica gel chromatography, using as solvent amixture of methylene chloride/methyl alcohol/2.5N NH₄ OH, 80:20:2. Thefractions containing the β-2-O-ethylglycoside dimethylaminoethylamide ofN-palmitoylneuraminic acid were gathered and evaporated under vacuum.The residue was dissolved in 60 ml of water. A stoichiometric amount ofmaleic acid was added, and the mixture was lyophilized. Yield: 80%.

Rf=0.11, chloroform/methyl alcohol, 70:30;

0.43, chloroform/methyl alcohol/2.5N NH₄ OH, 110:40:6.

EXAMPLE 32 β-2-ethylglycoside dimethylaminopropylamide ofN-dichloroacetyl neuraminic acid

4.34 g (10 mM) of β-2-O-ethylglycoside N-dichloro-acetylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methanol; 10.2 g (100mM) of dimethylamino-propylamine were added. The mixture was stirred for5 hours at 40° C. The solution was evaporated under vacuum and theresidue was purified by silica gel chromatography, using as solvent amixture of methylene chloride/methyl alcohol/water, 40:60:15. Thefractions containing the β-2-O-ethylglycoside dimethyl-aminopropylamideof N-dichloroacetylneuraminic acid were gathered and evaporated undervacuum. The residue was dissolved in 60 ml of water and lyophilized.Yield: 65%.

Rf=0.37, chloroform/methyl alcohol/2.5N NH₄ OH, 40:60:15.

EXAMPLE 33 β-2-O-ethylglycoside dimethylaminopropylamide ofN-dichloroacetylneuraminic acid (maleic acid salt)

4.34 g (10 mM) of the β-2-O-ethylglycoside ofN-dichloro-acetylneuraminic acid ethyl ester were solubilized in 50 mlof anhydrous methanol; 10.2 g (100 mM) of dimethylamino-propylamine wereadded. The mixture was stirred for 5 hours at 40° C. The solution wasevaporated under vacuum and the residue was purified by silica gelchromatography, using as solvent a mixture of methylene chloride/methylalcohol/water, 40:60:15. The fractions containing theβ-2-O-ethylglycoside dimethyl-aminopropylamide ofN-dichloroacetylneuraminic acid were gathered and evaporated undervacuum. The residue was dissolved in 60 ml of water. A stoichiometricamount of maleic acid was added, and the mixture was lyophilized. Yield:65%.

Rf=0.37, chloroform/methyl alcohol/2.5N NH₄ OH, 40:60:15.

EXAMPLE 34 β-2-O-ethylglycoside dimethylaminopropylamide of neuraminicacid

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylester, prepared according to Schauer and Buscher, Biochim. Biophys. Acta338, 369 (1974), were solubilized in 50 ml of anhydrous methanol; 10.2 g(100 mM) of dimethylamino-propylamine were added. The mixture wasstirred overnight at 40° C. The solution was evaporated under vacuum andthe residue was purified by reverse phase chromatography, using assupport Lichroprep RP 18 (Merck, Darmstadt, Germany) and as eluant amixture of methyl alcohol/water, 1:1. The fractions containing theβ-2-O-ethylglycoside dimethyl-aminopropylamide of neuraminic acid weregathered and evaporated under vacuum. The residue was dissolved in 50 mlof water and lyophilized. Yield: 60%.

Rf=0.1, chloroform/methyl alcohol/0.3% CaCl₂, 55:45:10.

EXAMPLE 35 β-2-O-ethylglycoside dimethylamide ofN-dichloroacetylneuraminic acid

4.34 g (10 mM) of the β-2-O-ethylglycoside ofN-dichloro-acetylneuraminic acid ethyl ester were solubilized in 50 mlof anhydrous methanol; 4.5 g (100 mM) of dimethylamine were added. Themixture was stirred overnight at 40° C. The solution was evaporatedunder vacuum and the residue was purified by silica gel chromatography,using as solvent a mixture of methylene chloride/methyl alcohol/water,80:10:1. The fractions containing the β-2-O-ethylglycosidedimethyl-amide of N-dichloroacetylneuraminic acid were gathered andevaporated under vacuum. The residue was crystallized from 60 ml ofmethanol and 300 ml of ethyl ether. Yield: 60%.

Rf=0.44, chloroform/methyl alcohol/2.5N NH₄ OH, 110:40:6.

EXAMPLE 36

α-2-O-ethylglycoside ethanolamide of N-palmitoyl-neuraminic acid

5.48 g (10 mM) of the α-2-O-ethylglycoside of N-palmitoylneuraminic acidethyl ester were solubilized in 50 ml of anhydrous methanol; 6.11 g (100mM) of ethanolamine were added. The mixture was stirred overnight at 35°C. The solution was evaporated under vacuum and the residue was purifiedby silica gel chromatography, using as solvent a mixture of methylenechloride/methyl alcohol, 90:10. The fractions containing theα-2-O-ethylglycoside ethanolamide of N-palmitoyl-neuraminic acid weregathered and evaporated under vacuum. The residue was dissolved in 60 mlof dioxane/water, 2:1, and lyophilized. Yield: 85%.

Rf=0.66, chloroform/methyl alcohol/H₂ O, 110:40:6.

EXAMPLE 37 β-2-O-ethylglycoside dimethylamide of neuraminic acid

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylester were solubilized in 50 ml of water and 11 ml (10 mM) of 1M NaOHwere added. The solution was maintained at 25° C. for 30 minutes. 2 mlof anhydrous Dowex 50x8 resin, H⁺ form, were added. The filteredsolution was evaporated under vacuum. The residue was solubilized in 50ml of anhydrous pyridine. 2.3 g (20 mM) of pyridinium chloride and 4.12g (20 mM) of N,N'-dicyclohexylcarbodiimide were added. The mixture wasstirred for 2 hours at 25° C. 4.5 g (100 mM) of diethylamine were addedand the reaction was conducted overnight at 25° C. The solution wasevaporated, and the residue was purified by silica gel chromatography,using as solvent a mixture of methylene chloride/methyl alcohol/2.5N NH₄OH, 110:40:6. The fractions containing the β-2-O-ethylglycosidedimethylamide of neuraminic acid were gathered and evaporated undervacuum. The residue was dissolved in 50 ml of water and lyophilized.Yield: 55%.

Rf=0.30, chloroform/methyl alcohol/H₂ O, 55:45:10.

EXAMPLE 38 β-2-O-ethylglycoside dimethylamide of N-caprylneuraminic acid

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylester were solubilized at 5° C. in 100 ml anhydrous methanol and 50 mlof anhydrous methylene chloride. 8.25 g (40 mM) ofN,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 3.44g (20 mM) of capric acid were added, and the mixture was stirredovernight at 5° C. After filtration, it was evaporated. The residue wassolubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1MNaOH were added. The solution was maintained at 25° C. for 30 minutes. 2ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. The filteredsolution was evaporated under vacuum. The residue was solubilized in 50ml of anhydrous pyridine. 2.3 g (20 mM) of pyridinium chloride and 4.12g (20 mM) of N,N'-dicyclohexylcarbodiimide were added. The mixture wasstirred for 2 hours at 25° C. 4.5 g (100 mM) of diethylamine were addedand the reaction was conducted overnight at 25° C. The solution wasevaporated, and the residue was purified by silica gel chromatography,using as solvent a mixture of methylene chloride/methyl alcohol/H₂ O,80:10:1. The fractions containing the β-2-O-ethylglycoside dimethylamideof N-caprylneuraminic acid were gathered and evaporated under vacuum.The residue was dissolved in 50 ml of acetone and precipitated in 20volumes of n-hexane. Yield: 65%.

Rf=0.48, chloroform/methyl alcohol/H₂ O, 80:10:1.

EXAMPLE 39 β-2-O-ethylglycoside dimethylamide of N-capryloyl-neuraminicacid

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylester were solubilized at 5° C. in 100 ml anhydrous methanol and 50 mlof anhydrous methylene chloride. 8.25 g (40 mM) ofN,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 2.88g (20 mM) of caprylic acid were added and the mixture was stirredovernight at 5° C. After filtration, it was evaporated. The residue wassolubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1MNaOH were added. The solution was maintained at 25° C. for 30 minutes. 2ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. The filteredsolution was evaporated under vacuum. The residue was solubilized in 50ml of anhydrous pyridine. 2.3 g (20 mM) of pyridinium chloride and 4.12g (20 mM) of N,N'-dicyclohexylcarbodiimide were added. The mixture wasstirred for 2 hours at 25° C. 4.5 g (100 mM) of diethylamine were addedand the reaction was conducted overnight at 25° C. The solution wasevaporated, and the residue was purified by silica gel chromatography,using as solvent a mixture of methylene chloride/methyl alcohol/H₂ O,80:10:1. The fractions containing the β-2-O-ethylglycoside dimethylamideof N-capryloylneuraminic acid were gathered and evaporated under vacuum.The residue was dissolved in 50 ml of acetone and precipitated in 20volumes of n-hexane. Yield: 65%.

Rf=0.61, chloroform/methyl alcohol/H₂ O, 80:20:2.

EXAMPLE 40 β-2-O-ethylglycoside dimethylamide of N-oleylneuraminic acid

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylesterwere solubilized at 5° C. in 100 ml of anhydrous methanol and 50 ml ofanhydrous methylene chloride. 8.25 g (40 mM) ofN,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 5.65g (20 mM) of oleic acid were added, and the mixture was stirredovernight at 5° C. After filtration, it was evaporated. The residue wassolubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1MNaOH were added. The solution was maintained at 25° C. for 30 minutes. 2ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. The filteredsolution was evaporated under vacuum. The residue was solubilized in 50ml of anhydrous pyridine. 2.3 g (20 mM) of pyridinium chloride and 4.12g (20 mM) of N,N'-dicyclohexylcarbodiimide were added. The mixture wasstirred for 2 hours at 25° C. 4.5 g (100 mM) of diethylamine were addedand the reaction was conducted overnight at 25° C. The solution wasevaporated, and the residue was purified by silica gel chromatography,using as solvent a mixture of methylene chloride/methyl alcohol, 9:1.The fractions containing the β-2-O-ethylglycoside dimethylamide ofN-oleylneuraminic acid were gathered and evaporated under vacuum. Theresidue was dissolved in 50 ml of tert-butanol and lyophilized. Yield:50%.

Rf=0.42, chloroform/methyl alcohol/H₂ O, 80:20:2.

EXAMPLE 41 β-2-O-ethylglycoside dimethylamide of N-valproyl-neuraminicacid

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylesterwere solubilized at 5° C. in 100 ml of anhydrous methanol and 50 ml ofanhydrous methylene chloride. 8.25 g (40 mM) ofN,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 2.88g (20 mM) of valproic acid were added and the mixture was stirredovernight at 5° C. After filtration, it was evaporated. The residue wassolubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1MNaOH were added. The solution was maintained at 25° C. for 30 minutes. 2ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. The filteredsolution was evaporated under vacuum. The residue was solubilized in 50ml of anhydrous pyridine. 2.3 g (20 mM) of pyridinium chloride and 4.12g (20 mM) of N,N'-dicyclohexylcarbodiimide were added. The mixture wasstirred for 2 hours at 25° C. 4.5 g (100 mM) of diethylamine were addedand the reaction was conducted overnight at 25° C. The solution wasevaporated, and the residue was purified by silica gel chromatography,using as solvent a mixture of methylene chloride/methyl alcohol/H₂ O,80:10:1. The fractions containing the β-2-O-ethylglycoside dimethylamideof N-valproylneuraminic acid were gathered and evaporated under vacuum.The residue was dissolved in 50 ml of acetone and precipitated in 20volumes of n-hexane. Yield: 50%.

Rf=0.60, chloroform/methyl alcohol/H₂ O, 80:20:2.

EXAMPLE 42 β-2-O-ethylglycoside dimethylamide ofN-phenylacetyl-neuraminic acid

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylester were solubilized at 5° C. in 100 ml of anhydrous methanol and 50ml of anhydrous methylene chloride. 8.25 g (40 mM) ofN,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 2.72g (20 mM) of phenylacetic acid were added, and the mixture was stirredovernight at 5° C. After filtration, it was evaporated. The residue wassolubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1MNaOH were added. The solution was maintained at 25° C. for 30 minutes. 2ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. The filteredsolution was evaporated under vacuum. The residue was solubilized in 50ml of anhydrous pyridine. 2.3 g (20 mM) of pyridinium chloride and 4.12g (20 mM) of N,N'-dicyclohexylcarbodiimide were added. The mixture wasstirred for 2 hours at 25° C. 4.5 g (100 mM) of diethylamine were added,and the reaction was conducted overnight at 25° C. The solution wasevaporated, and the residue was purified by silica gel chromatography,using as solvent a mixture of methylene chloride/methyl alcohol/H₂ O,80:10:1. The fractions containing the β-2-O-ethylglycoside dimethylamideof N-phenylacetylneuraminic acid were gathered and evaporated undervacuum. The residue was dissolved in 50 ml of acetone and precipitatedin 20 volumes of n-hexane. Yield: 65%.

Rf=0.43, chloroform/methyl alcohol/H₂ O, 80:20:2.

EXAMPLE 43 β-2-O-ethylglycoside dimethylamide of N-miristoyl-neuraminicacid

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylester were solubilized at 5° C. in 100 ml of anhydrous methanol and 50ml of anhydrous methylene chloride. 8.25 g (40 mM) ofN,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 4.57g (20 mM) of miristic acid were added, and the mixture was stirredovernight at 5° C. After filtration, it was evaporated. The residue wassolubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1MNaOH were added. The solution was maintained at 25° C. for 30 minutes. 2ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. The filteredsolution was evaporated under vacuum. The residue was solubilized in 50ml of anhydrous pyridine. 2.3 g (20 mM) of pyridinium chloride and 4.12g (20 mM) of N,N'-dicyclohexylcarbodiimide were added. The mixture wasstirred for 2 hours at 25° C. 4.5 g (100 mM) of diethylamine were addedand the reaction was conducted overnight at 25° C. The solution wasevaporated, and the residue was purified by silica gel chromatography,using as solvent a mixture of methylene chloride/methyl alcohol/H₂ O,80:10:1. The fractions containing the β-2-O-ethylglycoside dimethylamideof N-miristoylneuraminic acid were gathered and evaporated under vacuum.The residue was dissolved in 50 ml of acetone and precipitated in 20volumes of n-hexane. Yield: 60%.

Rf=0.56, chloroform/methyl alcohol/H₂ O, 80:20:2.

EXAMPLE 44 β-2-O-ethylglycoside dimethylamide of N-lauroyl-neuraminicacid

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylester were solubilized at 5° C. in 100 ml of anhydrous methanol and 50ml of anhydrous methylene chloride. 8.25 g (40 mM) ofN,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 4.57g (20 mM) of lauric acid were added, and the mixture was stirredovernight at 5° C. After filtration, it was evaporated. The residue wassolubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1MNaOH were added. The solution was maintained at 25° C. for 30 minutes. 2ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. The filteredsolution was evaporated under vacuum. The residue was solubilized in 50ml of anhydrous pyridine. 2.3 g (20 mM) of pyridinium chloride and 4.12g (20 mM) of N,N'-dicyclohexylcarbodiimide were added. The mixture wasstirred for 2 hours at 25° C. 4.5 g (100 mM) of diethylamine were addedand the reaction was conducted overnight at 25° C. The solution wasevaporated, and the residue was purified by silica gel chromatography,using as solvent a mixture of methylene chloride/methyl alcohol/H₂ O,80:10:1. The fractions containing the β-2-O-ethylglycoside dimethylamideof N-palmitoylneuraminic acid were gathered and evaporated under vacuum.The residue was dissolved in 50 ml of acetone and precipitated in 20volumes of n-hexane. Yield: 60%.

Rf=0.54, chloroform/methyl alcohol/H₂ O, 80:20:2.

EXAMPLE 45 β-2-O-ethylglycoside dimethylamide of N-nicotinoyl-neuraminicacid

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylester were solubilized at 5° C. in 100 ml of anhydrous methanol and 50ml of anhydrous methylene chloride. 8.25 g (40 mM) ofN,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 2.46g (20 mM) of nicotinic acid were added, and the mixture was stirredovernight at 5° C. After filtration, it was evaporated. The residue wassolubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1MNaOH were added. The solution was maintained at 25° C. for 30 minutes. 2ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. The filteredsolution was evaporated under vacuum. The residue was solubilized in 50ml of anhydrous pyridine. 2.3 g (20 mM) of pyridinium chloride and 4.12g (20 mM) of N,N'-dicyclohexylcarbodiimide were added. The mixture wasstirred for 2 hours at 25° C. 4.5 g (100 mM) of diethylamine were addedand the reaction was conducted overnight at 25° C. The solution wasevaporated, and the residue was purified by silica gel chromatography,using as solvent a mixture of methylene chloride/methyl alcohol/H₂ O,80:10:1. The fractions containing the β-2-O-ethylglycoside dimethylamideof N-nicotinoylneuraminic acid were gathered and evaporated undervacuum. The residue was dissolved in 50 ml of methanol and precipitatedin 20 volumes of tert-butyl-ether. Yield: 50%.

Rf=0.27, chloroform/methyl alcohol/H₂ O, 80:20:2.

EXAMPLE 46 β-2-O-ethylglycoside dimethylamide ofN-trimethoxy-benzoylneuraminic acid

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylester were solubilized at 5° C. in 100 ml of anhydrous methanol and 50ml of anhydrous methylene chloride. 8.25 g (40 mM) ofN,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 4.24g (20 mM) of trimethoxybenzoic acid were added, and the mixture wasstirred overnight at 5° C. After filtration, it was evaporated. Theresidue was solubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10mM) of 1M NaOH were added. The solution was maintained at 25° C. for 30minutes. 2 ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. Thefiltered solution was evaporated under vacuum. The residue wassolubilized in 50 ml of anhydrous pyridine. 2.3 g (20 mM) of pyridiniumchloride and 4.12 g (20 mM) of N,N'-dicyclohexylcarbodiimide were added.The mixture was stirred for 2 hours at 25° C. 4.5 g (100 mM) ofdiethylamine were added and the reaction was conducted overnight at 25°C. The solution was evaporated, and the residue was purified by silicagel chromatography, using as solvent a mixture of methylenechloride/methyl alcohol/H₂ O, 80:10:1. The fractions containing theβ-2-O-ethylglycoside dimethylamide of N-trimethoxbenzoylneuraminic acidwere gathered and evaporated under vacuum. The residue was dissolved in50 ml of methanol and precipitated in 20 volumes of tert-butyl-ether.Yield: 60%.

Rf=0.52, chloroform/methyl alcohol/H₂ O, 80:20:2.

EXAMPLE 47 β-2-O-ethylglycoside pyrrolidylamide ofN-palmitoyl-neuraminic acid

5.56 g (10 mM) of the β-2-O-ethylglycoside of N-palmitoylneuraminicacid, sodium salt, were solubilized in 50 ml of anhydrous pyridine. 2.3g (20 mM) of pyridinium chloride and 4.12 g (20 mM) ofN,N'-dicyclohexylcarbodiimide were added. The mixture was stirred for 2hours at 25° C. 7.17 g (100 mM) of pyrrolidine were added and thereaction was conducted overnight at 25° C. The solution was evaporated,and the residue was purified by silica gel chromatography, using assolvent a mixture of methylene chloride/methyl alcohol, 9:1. Thefractions containing the β-2-O-ethylglycoside pyrrolidylamide ofN-palmitoylneuraminic acid were gathered and evaporated under vacuum.The residue was dissolved in 50 ml of acetone and precipitated in 20volumes of n-hexane. Yield: 90%.

Rf=0.67, chloroform/methyl alcohol/H₂ O, 80:20:2.

EXAMPLE 48 β-2-O-ethylglycoside of N-palmitoylneuraminic acid ethylester

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylester were solubilized at 5° C. in 100 ml of anhydrous methanol and 50ml of anhydrous methylene chloride. 8.25 g (40 mM) ofN,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 5.12g (20 mM) of palmitic acid were added, and the mixture was stirredovernight at 5° C. After filtration, it was evaporated. The residue wassolubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1MNaOH were added. The solution was maintained at 25° C. for 30 minutes. 2ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. The filteredsolution was evaporated under vacuum. The residue was solubilized in 150ml of water and the product was extracted once with 300 ml chloroformand then twice with 150 ml of chloroform; the organic phases were washedthree times with 150 ml of water, gathered, and evaporated. The residuewas purified by silica gel chromatography, using as solvent a mixture ofmethylene chloride/methyl alcohol, 9:1. The fractions containing theβ-2-O-ethylglycoside of N-palmitoylneuraminic acid were gathered andevaporated under vacuum. The residue was crystallized from 100 ml oftert-butyl-ether. Yield: 85%.

Rf=0.44, methylene chloride/methanol, 90:10.

EXAMPLE 49 β-2-O-ethylglycoside of N-palmitoylneuraminic acid

5.65 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylester were solubilized in 100 ml of ethanol/water, 1:1. 11 ml (10 mM) of1M NaOH were added. The solution was maintained at 25° C. for 30minutes. 2 ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. Thefiltered solution was evaporated under vacuum. Yield: 95%.

Rf=0.14, methylene chloride/methyl alcohol/H₂ O, 110:10:6.

EXAMPLE 50 α-2-O-ethylglycoside of N-palmitoyl-neuraminic acid methylester

3.37 g (10 mM) of the α-2-O-ethylglycoside of neuraminic acid methylester were solubilized in 30 ml of 1M NaOH at 80° C., and maintainedunder stirring overnight. The solution was passed through a columncontaining 200 ml of Bio-Rex 70 H+ weakly basic resin, and thendessicated under vacuum. The residue was redissolved with 50 ml ofanhydrous methanol. 4.13 g (20 mM) of N,N'-dicyclohexylcarbodiimide and2.3 g of pyridinium chloride were added, and the mixture was stirred for1 hour. After filtration, it was evaporated. The residue was solubilizedat 5° C. in 100 ml of anhydrous methanol and 50 ml of anhydrousmethylene chloride 8.25 g (40 mM) of N,N'-dicyclohexylcarbodiimide, 2.0g (20 mM) of triethylamine, and 5.12 g (20 mM) of palmitic acid wereadded, and the mixture was stirred overnight at 5° C. After filtration,it was evaporated. The residue was solubilized in 150 ml of water andthe product was extracted once with 300 ml chloroform and then twicewith 150 ml of chloroform; the organic phases were washed three timeswith 150 ml of water, gathered, and evaporated. The residue was purifiedby silica gel chromatography, using as solvent a mixture of methylenechloride/methyl alcohol, 9:1. The fractions containing theα-2-O-ethylglycoside of N-palmitoylneuraminic methyl ester were gatheredand evaporated under vacuum. The residue was crystallized from 100 ml oftert-butyl-ether. Yield: 70%.

Rf=0.65, methylene chloride/methanol, 90:10.

EXAMPLE 51 α-2-O-ethylglycoside of N-palmitoylneuraminic acid, sodiumsalt

5.47 g (10 mM) of the α-2-O-ethylglycoside of neuraminic acid methylester were solubilized in 100 ml of methanol/water, 1:1. 11 ml (10 mM)of 1M NaOH were added. The solution was maintained at 25° C. for 30minutes. 2 ml of anhydrous Dowex 50x8 resin, H⁺ form, were added. Thefiltered solution was evaporated under vacuum. Yield: 95%.

Rf=0.25, chloroform/methyl alcohol/H₂ O, 110:10:6.

EXAMPLE 52 β-2-O-ethylglycoside of N-dichloroacetylneuraminic acid ethylester

3.23 g (10 mM) of the β-2-O-ethylglycoside of neuraminic acid ethylester were solubilized in 50 ml of anhydrous pyridine at 25° C.; 14.3 g(100 mM) of methyl dichloroacetate were added. The mixture was stirredfor 24 hours and then evaporated under vacuum. The residue was purifiedby silica gel chromatography, using as solvent a mixture of methylenechloride/methyl alcohol/H₂ O, 80:10:1. The fractions containing theβ-2-O-ethylglycoside of N-dichloroacetylneuraminic ethyl ester weregathered and evaporated under vacuum. The residue was crystallized froma mixture of 50 ml of methanol and 200 ml of ethyl ether. Yield: 85%.

Rf=0.49, chloroform/methanol, 80:20.

Biological Studies

The antineuronotoxic activities of the new amides of the neuraminicacids of the present invention are demonstrated by the followingexperimental studies conducted with the β-2-O-ethylglycoside of thedimethylamide of N-palmitoylneuraminic acid of the formula: ##STR4##identified as ND37.

EXAMPLE 53 Antineuronotoxic effect of ND37 in vitro on cerebellargranule cells: protective effect on exogenous glutamate inducedneurotoxicity Materials and Methods

Cell Cultures

Primary cell cultures of cerebellar granule Cells were prepared from 6day old Sprague-Dawley rats. Neurons were grown in 35 mm plates for11-13 days and maintained in a humid environment (95% air and 5% CO₂).Cultures (3×10⁶ cells/plate) are mainly formed by granule cells (95%)with a small amount (5%) of glial cells (Gallo V. et al.: Selectiverelease of glutamate from cerebellar granule cells differentiating inculture. Proc. Natl. Acad. Sci. USA 79, 7919-23, 1982). Glialproliferation was prevented by arabinofuranoside cytosine.

Derivative ND37 was solubilized at a concentration of 1×10⁻² M indimethylsulfoxide (1% DMSO) and then diluted at various concentrationsin Locke's solution (154 mM NaCl/1/5.6 mM KCl/3.6 mM NaHCO₃ /2.3 mMCaCl₂ /1 mM MgCl₂ /5.6 mM glucose/5 mM HEPES, pH 7.4). The followingconcentrations were tested: 5×10⁻⁶ M, 1×10⁻⁵ M, and 2×10⁻⁵ M.

Description of the Exogenous Glutamate Neurotoxicity Model

The cell culture medium was aspirated from the plates (and correctlymaintained). Plates were washed (3×2 ml) with Locke's solution, andsolutions (1.5 ml) containing the compound to be tested (concentrationsfrom 5×10⁻⁶ M to 2×10⁻⁵ M) were added and incubated for 2 hours in anincubator at 37° C. (5% CO₂).

Treated cells were washed (3×2 ml) with Locke's solution+10% fetal calfserum, then washed (3×2 ml) with Locke's solution without Mg⁺⁺. 100 μMglutamate (1.5 ml) in Locke's solution (-Mg⁺⁺) or culture medium(controls) were added. Incubation with glutamate was performed for 15minutes at room temperature (27° C.). Glutamate was removed, the plateswere washed with Locke's solution (2×2 ml), and then incubated inpresence of the starting medium (correctly maintained) for 24 hours at37° C. in an incubator (5% C₂ O). At the end of the incubation, cellularviability was assayed via quantification by the MTT colorimetric test(Mosmann T.: Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J. Immunol. Meth.65, 55: 63, 1983 and modified according to Skaper S. D. et al.: Death ofcultured hippocampal pyramidal neurons induced by pathologicalactivation of N-methyl-D-aspartate receptors is reduced bymonosialogangliosides. J. Pharm. Exp. Ter. 259,1 452-457, 1991). Dataare expressed as % survival. The significance was calculated accordingto the Dunnett test.

Results

The obtained results (Table 1) show that:

ND37 has a marked antineuronotoxic activity: the presence of the freecompound in the incubation medium during the exposure to the glutamatetoxin is not necessary. The neuroprotective effect of ND37 is very high:at a concentration of 1×10⁻⁵ M there is a protection of about 63%, andthe highest protection (about 83%) is reached at a concentration of2×10⁻⁵ M.

                  TABLE 1                                                         ______________________________________                                        Antineuronotoxic effect of ND37 in cerebellar granule                         cells: protective effect on exogenous glutamate induced                       neurotoxicity                                                                 Groups             MTT values  % survival                                     ______________________________________                                        1)  control                0.195 ± 0.016                                                                        100                                      2)  glutamate              0.075 ± 0.009                                                                        38 ± 5                                3)  glutamate + ND37                                                                           (5 × 10.sup.-6 M)                                                                 0.110 ± 0.015                                                                        37 ± 5                                                 (1 × 10.sup.-5 M)                                                                  0.123 ± 0.006*                                                                      63 ± 3                                                 (2 × 10.sup.-5 M)                                                                  0.326 ± 0.016*                                                                      83 ± 6                                ______________________________________                                         Significance (Dunnett's test)                                                 *p < 0.01 (vs group 2)                                                   

EXAMPLE 54 Antineuronotoxic effect of ND37 in vitro in cerebellargranule cells: protective effect on exogenous glutamate-inducedneurotoxicity during cotreatment of cells with the active compoundMaterials and Methods

Cell Cultures

Primary cerebellar granule cells were prepared from 8 day old rats(Zivic Miller, Pittsburgh, Pa, USA). Neurons were cultivated in 35 mmplates for 7-8 days and maintained in a humid environment (95% air and5% CO₂). Cultures (3×10⁶ cells/plate) are mainly formed by granule cells(95%) with a small amount (5%) of glial cells (Gallo V. et al.:Selective release of glutamate from cerebellar granule cellsdifferentiating in culture. Proc. Natl. Acad. Sci. USA 79, 7919-23,1982). Glial proliferation was prevented by arabinofuranoside cytosine.

Derivative ND37 was solubilized at a concentration of 1×10⁻² M indimethylsulfoxide (1% DMSO), and then diluted at various concentrationsin Locke's solution (154 mM NaCl/5.6 mM KCl/3.6 mM NaHCO₃ /2.3 mM CaCl₂/1 mM MgCl₂ /5.6 mM glucose/5 mM HEPES, pH 7.4). The followingconcentrations were tested: 5×10⁻⁶ M, 1×10⁻⁵ M, and 2×10⁵ M.

Description of the Exogenous Glutamate Neurotoxicity model

The cell culture medium was aspirated from the plates (and correctlymaintained). Plates were washed (3×2 ml) with Locke's solution withoutMg⁺⁺, and solutions (1.5 ml) containing the compound to be tested(concentrations from 5×10⁶ M to 2×10⁵ M) were added and incubated for 2hours in an incubator at 37° C. (5% CO₂).

Treated cells were washed (3×2 ml) with Locke's solution+10% fetal calfserum, then washed (3×2 ml) with Locke's solution without Mg⁺⁺. 1.5 mlof Locke's solution (-Mg⁺⁺) or 50 μM of glutamate±the test compound(concentrations between 1 and 4×10⁻⁵ M) in 1.5 ml of Locke's solution(-M⁺⁺) were added. The incubation was conducted for 15 minutes (37° C.).Glutamate and the compound were removed. The plates were washed withLocke's solution (2×2 ml), and then incubated in the presence of thestarting medium (correctly maintained) for 24 hours at 37° C. in anincubator (5% CO₂). At the end of the incubation, cellular viability wasassayed via quantification by the fluorescein diacetate (FDA) andpropidium iodide (PI) colorimetric test (Manev H. et al.: Glutamateinduced neuronal death in primary cultures of cerebellar granule cells:protection by synthetic derivatives of endogenous sphingolipids; J.Pharm. Exp. Ther. 252,1 419-427, 1990). Monolayers were washed withLocke's solution and stained for 3 minutes at 22° C. with a solutioncontaining 36 μM FDA and 7 μM of PI. The stained cells where immediatelyanalyzed using a standard fluorescence microscope for epiillumination(Vanox Olympus, 450 nm excitation, 520 nm emission). FDA, a non polarester, crosses cell membranes and is hydrolyzed by intracellularesterases with the consequent production of a yellow greenish color.Neuronal damage influences the FDA-induced color and allows thepermeation of PI, which is a polar compound capable of interacting withnuclear DNA, producing a brilliant red fluorescence.

After glutamate treatment, some neurons can degenerate and detach fromthe plates. The loss of cells was estimated by comparing the number ofintact or degenerated neurons in a well defined field, which wasphotographed before and after 24 hours after application of glutamate.The percent of surviving neurons in 4 representative fields(magnification 40×) of each monolayer was determined by a researcherunaware of the experimental conditions, evaluating the FDA/PI color, andcalculating it as follows: ##EQU1##

Data are expressed as % of surviving cells. Significance was calculatedusing the Dunnett's test.

Results

The data in Table 2 show that the neuroprotective effect of ND37 onglutamate toxicity is immediate, i.e., ND37 protects neurons at a doseof 10-40 μM, even if administered contemporaneously with the applicationof the toxin. This shows the rapid mode of action of ND37.

                  TABLE 2                                                         ______________________________________                                        Antineuronotoxic effect of ND37 during cotreatment of                         exogenous glutamate on cerebellar granule cells (pro-                         tective effect)                                                                                   % surviving cells N = 4                                   Groups              (average + s.e.) FDA/PI test                              ______________________________________                                        1)  control                 89 ± 7.0                                       2)  glutamte                22 ± 1.5                                       3)  glutamate + ND37                                                                           (1 × 10.sup.-5 M)                                                                  34 ± 2.0                                                        (2 × 10.sup.-5 M)                                                                   70 ± 6.0*                                                      (4 × 10.sup.-5 M)                                                                  88 ± 4.5                                       ______________________________________                                         Significance (Dunnett's test)                                                 *p < 0.01 (vs group 2)                                                   

EXAMPLE 55 In vivo effect of ND37 on cerebral damage induced byintracerebroventricular (icv) injection of N-methyl-D-aspartate inneonatal rats Materials and Methods

Description of the Model

Experiments were performed on 7 day old neonatal rats weighing ca. 13grams. At the 7th day, animals, after ether anesthesia, were lesioned byicv injection of 25 nmoles/μl of N-methyl-D-aspartate (NMDA), Sigma, St.Louis, Mo, USA, according to the method described by McDonald et al.(McDonald J. W. et al.: Neurotoxicity of N-methyl-D-aspartate ismarkedly enhanced in developing rat central nervous system. Brain Res.459, 200-203, 1988). The excitotoxin was solubilized in saline, and thepH was brought to 7.4 by adding 1N NaOH. The NMDA injection (25nmoles/μl) was performed slowly (2 minutes) at the level of the rightlateral ventricle utilizing a Hamilton syringe. Saline was administeredto control animals (1 μl icv).

The compound was administered subcutaneously (sc) after suspension in 1%DMSO (experiment No. 1) or in 0.5% tragacanth (experiment No. 2). Thecompound was tested at the following doses: 1-3-5 mg/kg sc.

The treatment was conducted performing two administrations:

1 hour before NMDA injection;

immediately after NMDA injection.

Experimental Groups

1. (n=8) saline (1 μl)+saline (1 μl)

2. (n=14) NMDA (25 nmoles μl)+saline (1 μl)

3. (n=15) NMDA (25 nmoles μl)+ND37 (1 mg/kg/ml)

4. (n=15) NMDA (25 nmoles μl)+ND37 (3 mg/kg/ml)

5. (n=15) NMDA (25 nmoles μl)+ND37 (5 mg/kg/ml)

The number of animals utilized in each experimental group (correspondingto the total number of animals, i.e., experiment 1+experiment 2,according to Table 3) is indicated in brackets.

Parameters

Animals were sacrificed on the 12th day (i.e., 5 days after NMDAinjection) for the evaluation of the in toto brain weight, defined inmg.

The statistical significance was evaluated using Dunnett's test.

Results

The obtained results (Table 3) show that:

treatment with ND37 is effective in reducing the brain damage induced bythe excitotoxin (evaluated as the lowering of total brain weight;

ND37 is significatively effective (p<0.01) at a dose of 1 mg/kg sc.

                  TABLE 3                                                         ______________________________________                                        Antineurotoxic effect of ND37 in vivo: evaluation of the                      protection of cerebral damage (total brain weight) induced                    by NMDA in neonatal rat                                                                              Doses   Total brain weight                             Groups     Compounds   (mg/kg) (mg)                                           ______________________________________                                        Experiment n.1 (vehicle = DMSO 1%)                                            1.  (n = 4) control                                                                          + saline          1017                                         2.  (n = 7) NMDA                                                                             + saline           841                                         3.  (n = 8) NMDA                                                                             + ND 37     1      960*                                        4.  (n = 8) NMDA                                                                             + ND 37     3      968*                                        5.  (n = 8) NMDA                                                                             + ND 37     5      959*                                        Experiment n.2                                                                1.  (n = 4) control                                                                          + saline          1017                                         2.  (n = 7) NMDA                                                                             + saline          >841                                         3.  (n = 7) NMDA                                                                             + ND 37     1     1019                                         4.  (n = 7) NMDA                                                                             + ND 37     3      977*                                        5.  (n = 7) NMDA                                                                             + ND 37     5      976*                                        ______________________________________                                         *p < 0.01 vs lesioned not treated (group 2)                                   (In all experiments the standard deviation is less than 5%).                  In brackets is the indication of the number of animals.                       Compounds under examination were administered at doses of 13-5 mg/kg sc 1     hours before NMDA injection (25 nmoles/1 ul) and immediately after NMDA.      Brain weight is expressed in mg.                                         

Modulatory in vitro effect of ND37 on release and/or uptake ofexcititatory negrotransmitter in cerebellar granule cells: determinationof glutamate and aspartate content in potassium induced depolarizationMaterials and Methods

Cell cultures

Primary cell cultures of cerebellar granule cells were prepared from 8day old Sprague-Dawley rats.

Neurons were grown in 35 mm plates for 11-13 days and maintained in ahumid environment (95% air and 5% CO₂). Cultures (3×10⁶ cells/plate) aremainly formed by granule cells (95%, with a small amount (5%) of glialcells (Gallo V. et al.: Selective release of glutamate from cerebellargranule cells differentiating in culture. Proc. Natl. Acad. Sci. USA 79,7919-23, 1982). Glial proliferation was prevented by arabinofuranosidecytosine.

Derivative ND37 was solubilized at a concentration of 10M indimethylsulfoxide (1% DMSO), and then diluted at various concentrations(0.1-1-10-20 μM) in Locke's solution (154 mM NaCl/5.6 mM KCl/3.6 mMNaHCO₃ /2.3 mM CaCl₂ /1 mM MgCl₂ /5.6mM glucose/5 mM HEPES, pH 7.4).

Description of Exogenous Glutamate Neurotoxicity Model

The cell culture medium was aspirated from the plates. Plates werewashed (3×2 ml) with Locke's solution, and solutions (750 μl) containingthe compound to be tested (concentrations from 0.1 to 20 μM) were addedand incubated for 2 hours in an incubator at 37° C. (5% CO₂) both in thepresence and absence of different depolarizing concentrations of KCl(between 5 and 50 mM). The incubation medium was gathered, filteredthrough a 0.2 μ filter, and processed (220 μl) for the analysis of theamino acid content.

Parameters

The glutamate and aspartate content (expressed as μM) was evaluated byHPLC (high pressure liquid chromatography), according to BidlingmeryerB. A. et al., J. Chromatogr. 336, 93-104, 1984.

Results

The obtained results show that ND37 is able to diminish in cerebellargranule cells, in a dose-dependent manner (Table 4), thepotassium-induced increase of glutamate and aspartate (Table 5). Thecompound, administered at doses up to 20 μM, did not modify the basallevels (controls) of extracellular glutamate and aspartate (Table 5),whereas it reduced by ca. 30% the extracellular glutamate induced by KClpotassium already at a dose of 0.1 μM (Table 4). ND 37 completelyabolished the glutamate increase induced by KCl in the culture medium ata dose of 20 μM (Table 5).

20 μM ND73 completely abolished the extracellular increase of glutamateand aspartate induced by KCl (15, 25, 30, and 50 mM). The extracellularvalues for glutamate and aspartate in cultures exposed to KCl in thepresence of ND37 were between 0.01 and 0.28% of the values obtainedwithout the compound (Table 5).

The possible mechanism of action of ND37 may reside in the releaseand/or uptake of endogenous compounds.

                  TABLE 4                                                         ______________________________________                                        Dose dependent inhibitory action of ND37 (0.1 - 1 - 10 -                      20 uM) on extracellular glutamate increase induced by KCl                     in primary cerebellar granule cells.                                                            Extracellular  % KCl                                        Groups            glutamate content (uM)                                                                       50 Mm                                        ______________________________________                                        1)  controls          0.04 ± 00                                            2)  KCl (50 mM)       17.40 ± 0.19                                                                              100                                      3)  KCl (50 mM) + ND37 (0.1 uM)                                                                     12.60 ± 0.57                                                                              72                                       4)  KCl (50 mM) + ND37 (1 uM)                                                                       13.30 ± 1.33                                                                              76                                       5)  KCl (50 mM) + ND37 (10 uM)                                                                       5.60 ± 2.37*                                                                             32                                       6)  KCl (50 mM) + ND37 (20 uM)                                                                       0.11 ± 0.09*                                                                             0.01                                     ______________________________________                                         Triplicate experiments (mean + s.e.)                                          *p < 0.01 vs. group 2 (Dunnett's test)                                   

                  TABLE 5                                                         ______________________________________                                        Effect of ND37 (20 uM) on extracellular glutamate and                         aspartate content in cerebellar granule cells in depo-                        larizing conditions induced by different concentrations                       of KCl (15-50 mM).                                                                        Extracell.                                                                              %       Extracell.                                                  glutamate corresp.                                                                              aspartate                                                                             % corresp.                              Groups      content (uM)                                                                            KCl     content (uM)                                                                          KCl                                     ______________________________________                                        1)  controls    0.354           0.020                                         2)  controls +  0.362           0.039                                             ND 37 (20 uM)                                                             3)  KCl (30 mM) 23.115    100   1.016   100                                       KCl (50 mM) +                                                                             0.318*    0.01  0.063   0.06                                      ND37 (20 uM)                                                              4)  KCl (50 mM) 10.863    100   0.621   100                                       KCl (50 mM) +                                                                             0.246*    0.02  0.078   0.13                                      ND37 (20 uM)                                                              5)  KCl (25 mM) 5.195     100   0.386   100                                       KCl (50 mM) +                                                                             0.330*    0.06  0.107   0.28                                      ND37 (20 uM)                                                              6)  KCl (15 mM) 2.454     100   0.484   100                                       KCl (50 mM) +                                                                             0.899*    0.34  0.046   0.09                                      ND37 (20 uM)                                                              ______________________________________                                         Triplicate experiments                                                        *p < 0.01 (Dunnet's test) vs corresponding group only treated with KCl (i     all experiments standard deviation less than 5%)                         

EXAMPLE 56 Electrophysiological characterization of neurons treated withND37: absence of an effect on glutamate-stimulated cationic channelsMaterials and Methods

Primary cell cultures of cerebellar granule cells were prepared from 8day old rats (Zivic Miller, Pittsburgh, Pa, USA).

Neurons were grown on 35 mm plates for 7-8 days and maintained in ahumid environment (95% air and 5% CO₂). Cultures (2.5×10⁶ cells/plate)were mainly formed by granule cells (95%), with a small amount (5%) ofglial cells (Gallo V. et al.: Selective release of glutamate fromcerebellar granule cells differentiating in culture. Proc. Natl. Acad.Sci. USA 79, 7919-23, 1982). Glial proliferation was prevented byarabinofuranoside cytosine.

Primary cell cultures of cortical neurons were prepared from one day oldneonatal mice (Zivic Miller, Pittsburgh, Pa., USA) (Bertolino andVicini: Mol. Pharmacol. 34, 98-103, 1988).

Cultures were grown on 35 mm plates, to which was added 10 μg/ml ofpoly-L-lysine, at a density of 5×10⁵ cortical neurons/plate. Cultureswere prepared on basal Eagle medium (Gibco) containing 10% fetal calfserum (Gibco), 25 mM KCl, 2 mM glutamine, and 100 μg/ml gentamycine.Glial proliferation was prevented by adding 1 μM arabinofuranosidecytosine 24 hours after seeding. Cortical neurons were cultivated for 3weeks.

Derivative ND37 was solubilized at a concentration of 1×10⁻² M indimethylsulfoxide (1% DMSO), and then diluted at various concentrationsin Locke's solution (-Mg⁺⁺) (154 mM NaCl/5.6 mM KCl/3.6 mM NaHCO₃ /2.3mM CaCl₂ /5.6 mM glucose/5 mM HEPES, pH 7.4). ND37 was analyzed atconcentrations of 20 and 30 μM.

Description of electrophysiological measurements of glutamate-relatedcationic channel activity cerebellar granule neurons and corticalneurons culture

Experiments were performed at room temperature utilizing the followingmedium 145 mM NaCl, 1 mM CaCl₂, 5 mM glucose, and 5 mM HEPES/NaOH at pH7.4, and patch pipettes, close to the preparation, containing 145 mMCsCl₂, 1 mM CaCl₂, 11 mM ethyleneglycol (β-aminoethyl ether) bisN,N'-tetraacetic acid, 10 mM HEPES/Cs(OH)₂ at pH 7.2. Plates werecontinually perfused at a flow rate of 1 ml/min.

a) "Sealed" registrations on whole cells (Hamill O. P et al., "Improvedpatch-clamp techniques for high-resolution current recording from cellsand cell-free membranes patches", Pfluegers Arch. 391, 85-100,1981) wereperformed on cerebellar granule cells or cortical neurons from neonatalrats and grown in cultures for 1 or 2 weeks, respectively.

Glutamate was released under pressure (2-6 psi) from glass micropipettes(with tips having a greater delivery capacity than that, 4-6 μM, ofpatch pipettes) on the cell bodies of voltage clamped neurons(maintenance voltage--40 mV).

b) The activation by glutamate of single channels was externallyrecorded on membrane portions prepared from cerebellar granule cellspretreated with ND37 for 30 minutes at 37° C. Micropipettes, filled (1μM) with glutamate, were positioned close to the membrane portions andwere also utilized in order to drip the neurotransmitter. Single channelcurrents were registered according to the method described by Bertoliniand Vicini ("Voltage-dependent block by strychnine ofN-methyl-D-aspartic acid-activated cationic channels in rat corticalneurons in culture", Mol. Pharmacol. 34, 98-103, 1988).

Registration on the whole cell

Glutamate (50 μM), able to activate currents directed inside, wasreleased by pressure on voltage-clamped cell bodies of cerebellar andcortical neurons. Because these experiments were performed in theabsence of Mg⁺⁺, the response to glutamate was probably mediated by theNMDA and non-NMDA glutamate receptor. The combined administration ofND37 (30 μM) and glutamate (50 μM), does not influence the controlresponse, which is not different from the controls (mean 200 pA atvoltage--50 mV) in the three different conditions in 3 cerebellar and 5cortical preparations.

Results

In order to evaluate the activities of ND37 on glutamate response, 10different experiments were performed. In cerebellar granule cellmembrane preparations, high conductivity, glutamate-activated (50 pS)cationic channels are prevalent. Pretreatment of cells for 30 minuteswith 20 μM ND37 does not influence the channel conductivity and thefrequency of their opening (Table 6). According to the single channelregistration, no kinetic and conductivity variations were observed afterND37 treatment. The inside currents activated by glutamate (50 μM) givencontemporaneously with ND37 (30 μM) were not different from thoseactivated only by glutamate.

                  TABLE 6                                                         ______________________________________                                        Effect of ND37 on glutamate controlled cationic channels                      activity in cerebellar granule neurons and cortical                           neurons in culture.                                                                           Single channel current registration                                           (cerebellar granule neurons)                                                    Channel                                                     Group             conductivity                                                                            Opening frequence                                 n = 10            (ps)      (opening/sec)                                     ______________________________________                                        1)  Glutamate (1 mM)  50        4.5 ± 2.9                                  2)  Glutamate + ND37 (20 uM)                                                                        50        4.1 ± 2.1                                  ______________________________________                                    

Conclusions

ND37, a compound protecting neurons from glutamate receptor-mediatedtoxicity, does not seem to produce this effect blocking the ionotropicglutamate receptors, as can be seen from the absence of effects ofcationic glutamate stimulated channels.

EXAMPLE 57

The neuritogenic activity of the new compounds of the present inventioncan be shown by experiments performed with the aforesaid compound ND37,and with the 2-ethylglycoside of N-palmitoyl-neuraminic aciddimethylaminopropylamide, which will be subsequently referred to asND35.

Materials and Methods

Cell Cultures

C1300 mouse neuroblastoma cells, Neuro-2a clone, (American Cell TypeCulture Collection, Bethesda, Md.) were grown at a density of 10,000cells/well in a culture medium containing Dulbecco's modified Eaglemedium (DMEM, Flow), 10% fetal calf serum (FCS, batch IP 02, Seromed),penicillin (100 units per ml, Irvine), and L-glutamine (2 mM, Sigma).Cells were incubated at 37° C. for 24 hours, and medium was removed andsubstituted with 350 μl of fresh medium plus compounds to be tested.

Tested Compounds and Their Solubilization

Compounds ND35 and ND37 were solubilized at a concentration of 1×10⁻² Min dimethylsulfoxide (1% DMSO). For the different compounds, progressivedilutions in culture medium were performed (concentrations from 1×10⁻⁵to 1×10⁻⁴ M).

Parameters

Neuritogenic activity (cell number with neurites, optical microscopy)

Culture plates were incubated with compounds to be tested and analyzedusing a phase contrast microscope (250×). Nine fields with prefixedcoordinates were chosen and photographed. Then the total number of cellsand those with neurites (length at least double the cell diameter) werecounted in blind in each photograph. The percentage of cells withneurites was determined after counting of at least 100 cells (Facci L.et al.: Promotion of neuritogenesis in mouse neuroblastoma cells byexogenous ganglioside GM1. J. Neurochem. Raven Press, New York,299-305,1984).

Results

The obtained results (Table 7) show that derivatives ND35 and ND37 bothinduce neuritogenesis in vitro. In particular, under the testedexperimental conditions, it was shown that:

the neuritogenic effect was already significant at a concentration of5×10⁻⁵ M (p<0.01), with the highest efficacy (about 48% of cells withneurites) at a concentration of 1×10⁻⁴ M.

                  TABLE 7                                                         ______________________________________                                        Neuritogenic effect of ND35 and ND37 in N2a neuroblastoma                     cells.                                                                        Compounds (concentrations)                                                                      Cells with neurities (%)                                    ______________________________________                                        Control               2 ± 3                                                ND37        (1 × 10.sup.-4)                                                                   48 ± 9*                                                          (5 × 10.sup.-5)                                                                   27 ± 5*                                                        (2.5 × 10.sup.-5)                                                                   8 ± 4                                                            (1 × 10.sup.-5)                                                                   5 ± 3                                                ND35        (1 × 10.sup.-4)                                                                   39 ± 7*                                                          (5 × 10.sup.-5)                                                                   28 ± 7*                                                        (2.5 × 10.sup.-5)                                                                   7 ± 3                                                            (1 × 10.sup.-5)                                                                   7 ± 2                                                ______________________________________                                         *p < 0.01 vs. control (Dunnett's test)                                   

CONCLUSIONS

The foregoing results show a good pharmacological profile of thecompounds according to the present invention. Their antineuronotoxic andmodulatory effects on extracellular levels of excititatoryneurotransmitter amino acids should be noted in particular.

Owing to their antineuronotoxic activity, the new derivatives ofneuraminic acid can be used in pathologies related to the excitotoxiceffect of excititatory amino acids. It has been shown that these aminoacids, e.g., glutamic acid and aspartic acid, besides their importantfunctions in several physiological processes such as, for example,synaptogenesis and plasticity, are involved in the ethiogenesis and/orevolution of different pathologies related to neuronal evolution and/ordeath. Although neuronal damage can have several causes, neuronaldysfunctions excite a cascade of cellular events, such as the activationof Ca⁺⁺ ion-dependent enzymes, the influx of Ca⁺⁺ ions, and theactivation of second messengers, which cause neuronal death. Damage tothe nervous system due to excititatory amino acids is present inischaemia, hypoxia, epilepsy, trauma, compressions, metabolicdisfunctions, aging, and toxic-infective and chronic neurodegenerativediseases, like Alzheimer's and Huntington's diseases.

Because of the modulatory effect of the new derivatives on the processesof release and/or uptake, and of the increase in the intracellularspace, of neurotransmitter amino acids, these new compounds havetherapeutic relevance in neuro-psychiatric disorders, where thepathological event derives from the imbalance of the aforesaidprocesses. Owing to the fact that the protective action of thederivatives of invention against the toxicity of excititatory aminoacids occurs through the activation of glutamate receptors, the use ofthese compounds does not have the disadvantages of other knownderivatives, which block these receptors (see Olney J. W. et al.:"Pathological changes induced in cerebrocortical neurons byphencyclidine and related drugs", Science 224, 1360-1362, 1989; Olney J.W. et al.: "NMDA antagonist neurotoxicity: mechanism and prevention,Science 254, 1515-1518, 1991).

Finally, the new compounds of the present invention, because of theirneuritogenic activity, are valuable in therapies related to the recoveryof nervous functions in pathologies associated with neuronal damage,like peripheral neuropathies.

PHARMACEUTICAL APPLICATIONS OF THE COMPOUNDS OF THE PRESENT INVENTION

Objects of the present invention also include pharmaceuticalpreparations having, as active ingredients, one or more of the newaforesaid derivatives and, particularly, those especially mentioned orthose described in the foregoing examples.

These pharmaceutical preparations can used for oral, rectal, parenteral,local or intradermic use. They can therefore be in solid or semisolidform, e.g., pills, tablets, gelatineous soft capsules, capsules, softgelatin suppositories, etc. For parenteral use, it is possible to useformulations for intramuscular, subcutaneous, or transdermic use, orsuitable for infusions or intravenous injections, and they can thereforebe prepared as solutions of active components or as a lyophilized powderof the active components, eventually to be added to one or moreexcipients or pharmaceutically acceptable solvents, which are usable forthe aforesaid purpose, and which are osmolar with physiological fluids.For local application, spray preparations, e.g,. nasal sprays, can beemployed, or ointments for topical use, or plasters for transdermaladministration can be used.

The preparations of the present invention can be used both in man andanimals. Preferably, they contain between about 0.01% and 10% of theactive components for solutions, sprays, ointments and creams, andbetween 1% and 100%, preferably between 5% and 50% of the activecomponent, for solid form preparations.

The dosage will vary according to the indication, the desired effect,and the route of administration. For therapeutic administration, or forprophylaxis by the parenteral route, the dosage varies preferentiallybetween 0.05 and 10 mg per kg body weight per day, and especiallybetween 0.05 and 2 mg per kg body weight per day.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirirt and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

We claim:
 1. A compound selected from the group consisting of(a) acarboxylic amide of a compound of formula I: ##STR5## wherein C-1 isamidated, and wherein Ac represents an acyl residue of an aliphatic,araliphatic, aromatic, alicyclic or heterocyclic carboxylic acid, havingat least 10 but not more than 24 carbons, (b) a 2-hydrocarbyl-glycosideof said amide, (c) a peracylated derivative of said amide, or of the2-hydrocarbyl glycoside of said amide wherein all to the hydroxyl groupsof formula I are acylated and the amide substituent on C-2 is in the αposition, (d) a basic addition salt of one of the foregoing compounds,and (e) an acidic addition salt of one of the foregoing compounds havingan acidic or basic function.
 2. A carboxylic amide according to claim 1,wherein said acyl group Ac is an acyl residue of a substituted ornon-substituted acid, said substituted acid having from one to threesubstituents selected from the group consisting of a halogen atom, afree, esterified, or etherified hydroxylic or mercapto group, a free oresterified carboxylic or sulfonic group, a free or esterified carboxylicor sulfonic group transformed into an amide, a free or aminogroup-substituted hydrocarbylic group, and a hydrocarbylic groupinterrupted by an --SO--, --SO₂ --, or phenylene group.
 3. A carboxylicamide according to claim 2, wherein said halogen atom is a memberselected from the group consisting of fluorine, chlorine and bromine. 4.A carboxylic amide according to claim 2, wherein said esterifiedhydroxylic or mercapto group is the esterified form of an aliphatic oraromatic acid which acid has not more than 8 carbon atoms.
 5. Acarboxylic amide according to claim 2, wherein said etherified hydroxyor mercapto group, or said esterified carboxylic or sulfonic group, isthe etherified or esterified form of an aliphatic alcohol, whichaliphatic alcohol, has not more than 8 carbon atoms, or of anaraliphatic alcohol, which araliphatic alcohol has one benzene ring andan alkylene of 1 or 2 carbon atoms.
 6. A carboxylic amide according toclaim 2, wherein said hydrocarbylic group which substitutes said aminogroup is an alkyl with at least 8 carbon atoms or an aralkyl with onebenzene ring and an alkylene of 1 or 2 carbon atoms.
 7. A carboxylicamide according to claim 2, wherein said acyl group Ac is saturated. 8.A carboxylic amide according to claim 1, wherein said acyl group Ac isunsaturated and contains only one double bond.
 9. A carboxylic amideaccording to claim 1, wherein said acyl group Ac is the acyl residue ofan acid selected from the group consisting of capric, undecilic,di-tert-butyl-acetic, lauric, tridecilic, myristic, pentadecilic,palmitic, margaric, stearic, arachic, behenic and lignoceric acid.
 10. Acarboxylic amide according to claim 1, wherein said acyl group Ac is theacyl residue of tryptophan.
 11. A carboxylic amide according to claim 1,wherein said acyl group Ac is the acyl residue of a peptide.
 12. Acarboxylic amide according to claim 1, wherein said acyl group Ac is anacyl residue of trimethoxybenzoic acid or diphenyl-O,O'-dicarbonic acid.13. A carboxylic amide according to claim 1, wherein said acyl group Acis an acyl residue of a heterocyclic acid selected from the groupconsisting of cinchoninic, lysergic, isolysergic, dihydrolysergic,2-bromo-lysergic, 2-bromo-dihydrolysergic, 1-methyl-lysergic,1-methyl-dihydro-lysergic, and 1-methyl-2-bromo lysergic.
 14. Acarboxylic amide according to claim 1, wherein the amido group of theamidated C-1 carboxyl group is --CONH₂, or is substituted with a primaryor secondary aliphatic, aromatic, araliphatic, alicyclic or heterocyclicamine having not more than 24 carbon atoms.
 15. A carboxylic amideaccording to claim 14, wherein said amido group is substituted with oneto three groups selected from the group consisting of a free, esterifiedor etherified hydroxylic or mercapto group; a halogen atom; a free,esterified, or amide-modified carboxylic or sulfonic group; a free aminogroup; and a hydrocarbyl-substituted amino group wherein saidhydrocarbyl group is interrupted with an --SO-- or --SO₂ -- group.
 16. Acarboxylic amide according to claim 15, wherein said esterified hydroxyor mercapto group is substituted with an aliphatic or aromatic acidhaving not more than 8 carbon atoms.
 17. A carboxylic amide according toclaim 15, wherein said esterified hydroxy or mercapto group, or saidesterified carboxylic or sulfonic group, is substituted with analiphatic alcohol having not more than 8 carbon atoms, or with anaraliphatic alcohol having only one benzene ring and an alkylene of 1 or2 carbon atoms.
 18. A carboxylic amide according to claim 15, whereinsaid hydrocarbyl group substituting said amino group is an alkyl havingnot more than 8 carbon atoms, or an aralkyl with only one benzene ringand an alkylene of 1 or 2 carbon atoms.
 19. A carboxylic amide accordingto claim 14, wherein said amine is an alkyl- or dialkylamine havingbetween 1 and 12 carbon atoms.
 20. A carboxylic amide according to claim14, wherein said amine is an alkylenamine having between 3 and 6 carbonatoms constituting the ring.
 21. A carboxylic amide according to claim14, wherein the amine is methylamine, ethylamine, propylamine,hexylamine, diethylamine, dimethylamine, diisopropylamine, dihexylaminepyrrolidine, piperidine, or azepine.
 22. A carboxylic amide according toclaim 14, wherein the amine is an aliphatic diamine.
 23. A carboxylicamide according to claim 22, wherein said diamine is selected from thegroup consisting of ethylenediamine, trimethylenediamine,tetramethylenediamine, pentamethylenediamine, hexamethylenediamine,piperazine, and N-alkyl or C-alkyl derivatives of said diamine havingfrom 1 to 4 carbons in the alkyl group.
 24. A carboxylic amide accordingto claim 14, wherein the amine is aminoethanol, aminopropanol,mercaptoethylamine, morpholine, tiomorpholine, and a C1-C4 alkylderivative of said amine.
 25. A carboxylic amide according to claim 14,wherein the amine is an amino acid selected from the group consisting ofvaline, leucine, phenylalanine, tryptophan, α-aminobutyric acid,β-aminobutyric acid, methionine, lysine, aspartic acid, glutamic acid,proline, and hydroxyproline.
 26. A carboxylic amide according to claim14, wherein the amine is a natural or synthetic peptide having not morethan 12 carbon atoms.
 27. A carboxylic amide according to claim 14,wherein the amine is a peptide.
 28. A carboxylic amide according toclaim 14, wherein the amine is selected from the group consisting ofphosphatidyl-ethanolamine, phosphatidylserine, sphingosine,dihydrosphingosine, psychosine, dihydropsychosine,phosphorylcholine-sphingosine, phosphorylcholine-dihydrosphingosine, andphytosphingosine.
 29. A carboxylic amide according to claim 14, whereinthe amine is either an aromatic amine having one aromatic ring, whereinsaid aromatic ring is non-substituted, or an aromatic amine substitutedwith one to three functional groups selected from the group consistingof halogen, hydroxy, methoxy, carboxy sulfonyl, and C1-C4 aliphatichydrocarbylic.
 30. A carboxylic amide according to claim 14, wherein theamine is a heterocyclic amine selected from the group consisting of apyrimidine base, a purine, ephedrine, tyramine, and adrenaline.
 31. Acarboxylic amide according to claim 1, wherein the aglycon of the2-hydrocarbyl-glycoside is substituted with an aliphatic alcohol havingnot more than 12 carbon atoms.
 32. A carboxylic amide according to claim1, wherein the aglycon of the 2-hydrocarbyl-glycoside is substitutedwith an araliphatic alcohol having one benzene ring, wherein saidbenzene ring is non-substituted, or a benzene ring substituted with 1-3C1-C4 alkyl groups, and having not more than 4 carbon atoms in thealiphatic chain.
 33. A carboxylic amide according to claim 1, whereinthe aglycon of the 2-hydrocarbyl-glycoside is substituted with analicyclic alcohol or an aliphatic-alicyclic alcohol having only onecycloaliphatic ring and not more than 14 carbon atoms.
 34. A carboxylicamide according to claim 1, wherein the aglycon of the2-hydrocarbyl-glycoside is substituted with a heterocyclic alcoholhaving not more than 12 carbon atoms, and only one heterocyclic ringcontaining 1 or 2 heteroatoms selected from the group consisting of--NH--, --O--, and --S--.
 35. A carboxylic amide according to claim 1,wherein the aglycon of the 2-hydrocarbyl-glycoside is substituted with acorticosteroid steroid alcohol.
 36. A carboxylic amide according toclaim 1, wherein in the peracylated derivatives, the acyl groups areacyl residues of derived from aliphatic acids having not more than 10carbon atoms.
 37. A carboxylic amide according to claim 1, wherein inthe peracylated derivatives, the acyl groups are acyl residues ofaromatic acids having only one benzene ring.
 38. A compound which is theβ-2-O-ethylglycoside of N-palmitoyl-neuraminic acid.
 39. A compoundwhich is the β-2-O-ethylglycoside dimethylamide ofN-palmitoyl-neuraminic acid.
 40. A compound which is theβ-2-O-ethylglycoside L-alanyl-D-isoglutaminylamide ofN-palmitoylneuraminic acid.
 41. A compound selected from the groupconsisting of(a) a peracylated derivative of a carboxylic ester of thecompound of the following formula: ##STR6## wherein C-1 is amidated, andwherein Ac represents an acyl residue of an aliphatic, araliphatic,aromatic, alicyclic, or heterocyclic carboxylic acid, and (b) a basic oracidic addition salt of said peracylated derivative.
 42. A process forpreparing a peracylated derivative of a carboxylic amide ofN-acylneuraminic acid of claim 1 using a neuraminic acid having a freeamino group as a starting material, comprising introducing in a stepwisemanner,(a) the C-1 amide function, and (b) the acyl groups onto the freeamino group and each of the hydroxy groups of the compound of formula I.43. The process according to claim 42, further comprising forming a saltof said carboxylic amide.
 44. The process according to claim 42, whereinthe free amino group of a neuraminic acid starting material is acylatedwith the desired acid.
 45. A process for preparing aperacylated-derivative of a carboxylic ester or salt thereof accordingto claim 41, comprising introducing into the compound of formula I, in astepwise manner, (a) the ester function onto the carboxylic group and(b) the acyl groups onto each of the hydroxy groups.
 46. The processaccording to claim 42, further comprising introducing a 2-glycosidicgroup into the neuraminic acid starting material.
 47. The processaccording to claim 46, further comprising forming salts of said amide.48. A pharmaceutical composition, comprising a compound according toclaim 1 as active ingredient, and a pharmaceutically acceptable carrier.49. A method of reducing glutamate or aspartate induced neurotoxicitycomprising:exposing cells to a compound of claim 1 at a concentrationsufficient to prevent or reduce neurotoxicity induced by glutamate oraspartate-derived excititatory amino acids.
 50. A method of inducingneuritogenesis comprisingexposing cells to a compound of claim 1 at aconcentration sufficient for inducing neuritogenesis.